Method for detection of 4-hydroxybutyric acid and its precursor(s) in fluids

ABSTRACT

A method for determining the presence or amount of gamma-hydroxybutyrate or precursors in a sample, said method comprising contacting said sample with an indicator which specifically binds to gamma-hydroxybutyrate or precursors to form an indicatorcomplex; and, measuring said indicatorcomplex to determine the presence or amount of said gamma-hydroxybutyrate or precursors in said sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a division of application Ser. No. 09/607,026, Filed Jun.29, 2000, now U.S. Pat. No. 6,617,123 granted Sep. 9, 2003.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] As the use of illicit drugs in this country has increased, publicconcern over the problems associated with its effects has grown into amajor concern. This concern has led to workplace drug testing in orderto identify, treat, and remove active drug users from the workforce.This trend started in the military, and spread rapidly to lawenforcement and any “safety-sensitive” private sector jobs such asairline pilots, truck drivers, and active crewmembers of publictransportation. These initial strides into drug testing in the workplacerevealed the obtrusive incursion of drug use and abuse in the dailylives of a significant portion of Americans. Further research indicatedthe staggering costs to public and private industry in terms of lostproductivity, increased health care costs, and human suffering and deathdue to this scourge of drug abuse. As a result, drug testing has rapidlyspread to all areas of the public and private sector. The vast majorityof workplace drug testing has taken the form of urine testing, becauseof ease of collection, low cost, and effective indication of recent druguse. Other forms of testing include analysis of blood, saliva, sweat,and hair.

[0004] Gamma-hydroxybutyrate (4-hydroxybutyrate, 4-hydroxybutyric acid,Gamma-hydroxybutyric acid, 4-hydroxybutyric acid sodium salt, GHB) wasfirst used for anesthetic purposes in 1961, because it was unpredictableand caused adverse effects, its use was discontinued. Later, GHB wasused by body builders for muscle building and weight control. Presently,the U.S. DEA (Drug Enforcement Agency) is investigating GHB to see if itshould be a controlled substance. The U.S. FDA (Food and DrugAdministration) list GHB as an unapproved drug except forinvestigational use in the treatment of narcolepsy. Common names for GHBare Scoop, Georgia Home Boy, Grievous Bodily Harm, Liquid Ecstasy, andCherry Meth among others. Its precursor GBL (gamma-butyrolactone) isused as a GHB substitute and once ingested rapidly converts to GHB. Thepharmacological effects of both GHB and GBL are similar and the range ofanalgesic effect (euphoria) are similar.

[0005] In the 1990's GHB has become a popular drug of abuse on collegecampuses, bars, and dance clubs and is called the “date rape” drug. Theabuse of GHB has enormous sociological and economic impact on oursociety. A typical “date rape” scenario is as follows: The victim(s),usually women, are in a bar, they drink a beverage that has been lacedwith GHB by a rapist, the victim then becomes catatonic and is usuallyagreeable with anyone and everyone around them. They can becomeunconscious and then of course are susceptible to the rapist desires.Later, the victim(s) wakes up completely disoriented, naked and robbed.This type of horrific tragedy is occurring on a daily basis. The victimsof “date rape” are not only exposed to the physical assault of rape androbbery, but to the contraction of diseases such as AIDS and STD's. Thedamage caused and the consequences of such occurrences are immeasurable.

[0006] Accordingly, a need exists for providing an easy and convenientmanner by which to make a determination of the presence of GHB in urine,in a beverage, or other biological fluids or liquids. A further needexists for a convenient manner by which such determinations may be madeby using rapid analysis manual techniques (such as a dipstick or lateralflow devices) and automated techniques that will advance the artsignificantly. And, the most important need is for a device that woulddetect GHB using just a single assay that does not require an extractionprocess or lactone conversion. This would be a marked advancement in theart and would result in the savings of millions of dollars to the drugtesting laboratories required to perform GC (gas chromatography) or GCMS(gas chromatography mass spectrometry) testing for GHB and obviouslythis savings would be passed on to the end user (the businesses whichinitially request drug's of abuse assays on perspective and currentemployees). To explain further, the drug-testing laboratory wouldnormally perform GC (gas chromatography) or GCMS (gas chromatographymass spectrometry) assay for GHB. The necessary time to perform theseassays is burdensome to the laboratory through cost for tech time,reagents, and turnaround time to mention a few. The alternative to thiswould be the significant advancement in the art that the present deviceoffers which is the capability to detect GHB without lengthy extractionprocesses of the current art with a single assay.

[0007] 2. Description of the Related Art

[0008] This invention is in the field of toxicology. More specifically,this invention provides test strips (i.e. dry chemistry dipsticks, oron-site test modules utilizing thin layer chromatography in a lateralflow format, or other similar technology to the test strip) and liquidchemistry reagents for use in the detection of GHB with a single assayin aqueous fluids to include but not limited to urine, saliva, serum,blood, sweat extracts, liquid homogenates of hair and liquids such asbeverages, soft drinks, mixed drinks to included alcohol, etc.

[0009] It is known that the polarity and small molecular size of the GHBmolecule and the lack of detectability of the GHB by UV (ultra violet),chromatographic, and spectrophotometric means complicates detection. GHBis relatively unstable and will form the GHB lactone derivative whenheated and under acidic conditions. GHB can cause euphoria at less than50 ug/mL to marked central depression, sleep, coma and death. Currently,all of the methods to detect GHB use and/or solid-phase extraction,liquid-liquid extraction, silyl-derivatization, and then GCMS. Thesemethods mentioned are very time consuming, expensive (costing thelaboratories, companies ordering drug testing, and general publicmillions of dollars per year), and labor intensive. The GC-MS, assay istypically performed to verify the urines that screen positive for drugsof abuse. The GC-MS analysis costs 100 times as much as the initialscreen ($100 vs $1). Every additional unnecessary GC-MS performed drivesup the overall cost of drug testing.

[0010] The novel invention described herein describes a method todetermine the presence or absence of GHB and its precursor(s) in urineor other fluids by liquid and dry chemistry test means which has notbeen taught prior to the present art. It should be noted that GHB is notnormally found in urine.

[0011] There are no published, taught, or even mentioned methods of thepresent arts technology to detect GHB or GBL in urine by the presentarts techniques.

[0012] Again, a thorough search of patents and research revealed norelative art (i.e., prior art) with any correlation to this technology.The art of testing for GHB or GBL in urine or other fluids as previouslydelineated in the literature describe various techniques includingmethods for solid-phase extraction, liquid-liquid extraction,silyl-derivatization, then GCMS. No reference, however, has describedthis new art as delineated here. The previous art will be enumeratedhere to further illustrate the unique advancement in the field of GHBand GBL detection. It has been acknowledged in the art that randomurinary sample matrices are very complex, and consist of many urinaryconstituents, which create strong buffering, and interference problems(e.g. cannibal like enzymes such as protease). In addition, diseasestates will significantly impact the nature of urinary contents. Urineis also the repository of all of the body's waste products includingexcess parent nutrients, vitamins, drugs, and their metabolites. Thesewaste chemicals vary from person to person and significantly contributeto the individual uniqueness that makes assay design for urinaryconstituents more difficult than any other body fluid. All of thesefactors impact an assay's ability to obtain acceptable precision andaccuracy. The ability of an assay to analyze a biological liquid such assaliva, therefore, rarely ever translates to an effective assay forurine. Therefore the present invention's ability to effectively copewith random urine samples and biological fluids or other fluids such asa beverage(s) makes it unique.

[0013] Patent, U.S. Pat. No. 3,603,957, discloses the use of assay teststrips, but fails to teach a method for the determination of GHB or GBLof a test sample submitted for drugs of abuse testing. It also doesn'tteach a method to determine the presence or absence of any substancesuch as GHB or GBL. The patent doesn't teach the use of the presentart's reaction formula to dry chemistry format called a dipstick orlateral flow technology that not only is completely novel, but preventscross contamination between test pads typically found on a test strip(dipstick). In addition, this patent also failed to mention any methodsfor determination of GHB or GBL by dipstick, lateral flow, colorimetric,liquid reagent (automated) or other suitable means.

[0014] Another patent, U.S. Pat. No. 4,351,899, discloses the use ofassaying metabolic acids specifically beta-hydroxybutyric acid and/orlactic acid, but fails to teach a method for the determination of GHB orGBL in a sample of fluid. The patent doesn't teach the use of a drychemistry format utilizing lateral flow and/or a lateral flow hybridmethod or any use for automated analyzers and has no bearing on thepresent invention. There is no similarity between beta-hydroxybutyricacid and GHB and/or GBL. GHB is an analgesic drug which can induce comasand death. GHB could not be manufactured from beta-hydroxybutyrate.Beta-hydroxybutyrate is a by-product of human metabolism, it is not adrug, and cannot provide or cause an analgesic effect.

[0015] Another patent, U.S. Pat. No. 4,622,296, discloses a process formeasuring the activity of a dehydrogenase or the amount of substratereacted or enzyme such as dehydrogenase. This assay has no bearing withregards to the present art. It makes no mention of GHB or GBL and failsto teach a method for the determination of GHB or GBL in a sample offluid. The patent doesn't teach the use of a dry chemistry formatutilizing, dipsticks, lateral flow and/or a lateral flow hybrid methodor any use for automated analyzers and has no bearing on the presentinvention. There is no similarity between dehydrogenase and GHB and/orGBL. GHB is an analgesic drug which can induce comas and death. GHBcould not be manufactured from a dehydrogenase. Dehydrogenase is anenzyme not GHB (a drug) or GBL a by-product of GHB metabolism.Dehydrogenase is not a drug, and cannot provide or cause an analgesiceffect.

[0016] Another patent, U.S. Pat. No. 5,912,139, discloses a method (teststrip) for measuring the activity of a dehydrogenase or the amount ofsubstrate reacted. This assay has no bearing with regards to the presentart. It makes no mention of GHB or GBL and fails to teach a method forthe determination of GHB or GBL in a sample of fluid. The patent doesn'tteach the use of a dry chemistry format utilizing lateral flow and/or alateral flow hybrid method or any use for automated analyzers and has nobearing on the present invention. There is no similarity between adehydrogenase and GHB and/or GBL. GHB is an analgesic drug which caninduce comas and death. GHB could not be manufactured fromdehydrogenase. Dehydrogenase id an enzyme and is not a drug, and cannotprovide or cause an analgesic effect.

[0017] Another patent, U.S. Pat. No. 5,624,813, discloses a method formeasuring the activity of a NAD(P)-linked dehydrogenase reactions. Thismethod has no bearing with regards to the present art. It makes nomention of GHB or GBL and fails to teach a method for the determinationof GHB or GBL in a sample of fluid. The patent doesn't teach the use ofa dry chemistry format utilizing lateral flow and/or a lateral flowhybrid method or any use for automated analyzers and has no bearing onthe present invention. There is no similarity between a NAD(P)-linkeddehydrogenase and GHB and/or GBL. GHB is an analgesic drug which caninduce comas and death. GHB could not be manufactured fromdehydrogenase. Dehydrogenase is an enzyme and is not a drug, and cannotprovide or cause an analgesic effect.

[0018] Another patent, EU #0 291 194, discloses a method for pregnancytesting. This method has no bearing with regards to the present art. Itmakes no mention of GHB or GBL and fails to teach a method for thedetermination of GHB or GBL in a sample of fluid. The patent doesn'tteach the use of a dry chemistry format utilizing lateral flow and/or alateral flow hybrid method or any use for automated analyzers and has nobearing on the present invention. There is no similarity betweenpregnancy testing and GHB and/or GBL. GHB is an analgesic drug which caninduce comas and death. GHB could not be manufactured from any pregnancytest or any teaching of any pregnancy test. A pregnancy is a method todetermine if an individual is pregnant. This cannot provide a method forthe detection of GHB or GBL and is not a drug, and cannot provide orcause an analgesic effect.

[0019] Another patent, U.S. Pat. No. 5,032,506, discloses a method formeasuring the activity of a NAD(P)H, HAD(P)-linked dehydrogenasereactions. This method has no bearing with regards to the present art.It makes no mention of GHB or GBL and fails to teach a method for thedetermination of GHB or GBL in a sample of fluid. The patent doesn'tteach the use of a dry chemistry format utilizing lateral flow and/or alateral flow hybrid method or any use for automated analyzers and has nobearing on the present invention. There is no similarity between aNAD-linked dehydrogenase and GHB and/or GBL. GHB is an analgesic drugwhich can induce comas and death. GHB could not be manufactured fromdehydrogenase. Dehydrogenase is an enzyme and is not a drug, and cannotprovide or cause an analgesic effect.

[0020] Another patent, EU #0 226 427, discloses the method ofsimultaneously inspecting quality of animal milk and health of theanimal secreting the milk and an indicator for beta-hydroxybutyric acid,but fails to teach a method for the determination of GHB or GBL in asample of fluid. The patent doesn't teach the use of a dry chemistrymethods or techniques or format utilizing lateral flow and/or a lateralflow hybrid method or any use for automated analyzers and has no bearingon the present invention. There is no similarity betweenbeta-hydroxybutyric acid and GHB and/or GBL. GHB is an analgesic drugwhich can induce comas and death. GHB could not be manufactured frombeta-hydroxybutyrate. Beta-hydroxybutyrate is a by-product of humanmetabolism, it is not a drug, and cannot provide or cause an analgesiceffect

[0021] Another patent, U.S. Pat. No. 4,301,115, discloses the use ofassay test strips, and the ability of the assay strips to resist crosscontamination between reactant areas (chemically impregnated test pads),but fails to teach a method for the determination of GHB or GBL in asample of fluid. The patent doesn't teach the use of the dry chemistryformat utilizing either a dipstick or lateral flow device, liquidreagent (automated) method or mention any methods for determination ofGHB or GBL.

[0022] Another patent, U.S. Pat. No. 5,447,837, does mention the use ofassay test strips but again fails to disclose a method for thedetermination of GHB or GBL. This is a method for detection of anantigenic substance in human, biological samples. This patent also failsto mention the use of a reaction formula that is adaptable to the drychemistry format utilizing either a dipstick or lateral flow device. Italso doesn't teach a method to determine the presence or absence of anysubstance such as GHB or GBL. In addition, this patent also failed tomention any methods for determination of GHB or GBL by dry chemistry,liquid chemistry, colorimetric, or other suitable means.

[0023] Published literature and the prior art describes techniques suchas ELISA that have been used to determine the presence of drugs ofabuse, but these technologies have no relevant bearing on the presentdevice. Previously taught technologies include measurement of GHB or GBLusing GC or GCMS techniques and the required extraction proceduresinherent (required) prior to analysis by GC or GCMS.

[0024] Therefore, in a nutshell, the present device provides an absolutenovel approach to GHB or GBL testing and lateral flow testing using drychemistry test pads and automated liquid reagent testing.

[0025] Not surprisingly, it is known and is illustrated here that agreat need exists in the field of drug testing for rapid, economical,and effective method for the detection of GHB or GBL on samplessubmitted for testing, whether liquid chemistry and/or dry chemistrymethodology using dipsticks or lateral flow test devices (for single useand for on-site collections). The present invention does detect GHB orGBL effectively with a single assay and therefore and accordingly, thepresent device provides an easy and convenient manner by which to make adetermination of the presence or absence of GHB or GBL in a fluid. Thepresent art's use of lateral flow also enables the removal of anyinterference of any cross over of reagents or fluid from one test pad toanother which is one of the exclusive problems with dipsticks.

[0026] It is clear that a need exists for a convenient manner by which adetermination of GHB or GBL can be made utilizing a rapid automatedanalysis utilizing a liquid reagent format of the present device ormanual analysis in the form of dry chemistry (dipstick) and/or lateralflow test devices. These and other advances in the current state of theart will become evident in view of the present specification and claims.

SUMMARY OF THE INVENTION

[0027] Briefly stated, the present invention relates to the test devicesfor detecting the presence of GHB and or its precursor GBL(gamma-butyrolactone) in a liquid test sample and the methods for makingsaid devices. GBL is also used as a GHB substitute. Once ingested, GHLis rapidly converted to GHB. GHB can also be converted to GBL by acidcatalysis. This invention is in the field of drug testing. Morespecifically, this invention provides dry chemistry test strips (i.e.dipsticks, or dry chemistry and lateral flow [thin layer chromatography]test means) or automated or manual liquid reagent means for use in thedetection of GHB or GBL in biological samples (e.g. urine, blood, serum,saliva, sweat extracts, and hair homogenates) or other fluids such asbeverages, water, soft drinks, alcoholic drinks to name a few. Thisinvention achieves this goal by measuring the presence of GHB or GBL ina test sample. And, this invention provides a unique method forpreventing cross contamination between test pads (reactant areas) ondipsticks by the present inventions use of the dipstick test pad andlateral flow device technology. This invention provides a previouslyunavailable dry chemistry or liquid chemistry method for determining GHBor GBL presence in a test sample by measuring the presence of GHB orGBL.

[0028] The present invention encompasses a method that can utilizeseveral different techniques. The techniques would employ the manualmethod using dry chemistry dipsticks and the method of combining drychemistry dipstick reactant areas (test pads) with lateral flow thinlayer chromatography or the method of using a liquid reagent that iscompatible with automated analyzers that provide high speed quantitativeanalysis which would be much less labor intensive than the manualmethods providing a savings in time and money. The widespread utility ofthe present art also provides the drug testing laboratory,over-the-counter user, individual, police agency, drug testingcollection site (where the urine is actually collected), or other usersthe choice of using the dry chemistry (manual) or automated liquid meanswhich ever method best suits their situation or needs.

[0029] The present arts technique utilizes two dry chemistry techniques,one is dipsticks, which is a carrier dependent, rapid test that usesabsorbent medium such as paper, which have been impregnated with achemical formulation to detect adulteration. After dipping one(dipstick) into a liquid test sample, a reaction takes place. Saidresulting reaction will yield a color change indicating a positive ornegative result (i.e. presence or absence of GHB or GBL). The othertechnique is the use of lateral flow in combination with a dry chemicaltest pad. The lateral flow device is a rapid test that uses absorbentmedium such as paper, which has been impregnated with the chemicalformulation to detect GHB or GBL. The paper, after impregnation, is thenplaced on a lateral flow medium, such as nitrocellulose paper, glassfiber paper, or other suitable wicking material that will deliver thetest sample to the impregnated paper. The lateral flow device works bydipping one end of the lateral flow device (LFD) into a sample (urinefor example). The urine migrates up (along) the paper (or absorbentmaterial) to the reactive sites (test paper) containing reagents(reactive ingredients). The urine constituents react with the assayreagents during the migration process and yield visible results. Theurine can also be droppered onto the LFD and the sample will thenmigrate along the paper.

[0030] The ease of use and rapid results obtained by the present art'smethodology illustrate the unique utility of this testing technique. Inaddition, very little technical expertise is required to perform thistype of assay (no instrumentation required). Furthermore, the earlydetection of GHB or GBL facilitates the prevention of the “date rape”syndrome. This novel concept for GHB or GBL monitoring provides anenormous savings of time and money. The present arts method(s) of GHB orGBL testing utilizing these techniques are currently not available andhave never been taught.

[0031] An important aspect of GHB or GBL testing in fluids is thesensitivity of the test method. Both techniques taught here have aneffective sensitivity range comparable to the GC-MS target range. Thesensitivity can also be adjusted to indicate a large amount present aswould be the case if the sample tested were a soft drink spiked with GHBor GBL. Obviously, the amount in a sample directly spiked with GHB orGBL would be much higher in concentration than the amounts found inurine. The dipstick test and LFD dipstick hybrid (to be known as the LFDhybrid) have a quantitative to qualitative assay range. The results areevaluated via one of following categories: negative, positive, orquantitatively.

[0032] The present arts technique also utilizes a liquid chemistry testmeans that allows for rapid analysis via an automated analyzer that canyield high-speed quantitative results. This will result in rapid testresults, improved accuracy, lowered labor cost, and better turn aroundon a high volume of test. This automated method is only limited by thespeed of the automated analyzer. Some analyzers currently on the marketare capable of over 10,000 test per a hour. The ability of the presentart to perform a single assay on a high speed automated analyzer that iscapable of detecting GHB or GBL has never been present or taught in theprior art.

[0033] It is currently known in the art that enzyme and antigen/antibodyreaction kinetics are related to the rate of change in analytical,biological systems. The variables that affect this rate of changeinclude concentration of reactants and product, temperature, pH, ionicstrength, buffer strength, and other parameters. The present art'sinnate and unique ability enables it to determine the presence of GHB orGBL in fluids. As it is known in the art urine is a very complex matrixand the measurement of GHB or GBL in fluids such as urine has to takeinto account many factors, which will affect the assay.

[0034] The composition of the formulation to be applied to the drychemistry dipstick, LFD hybrid and liquid chemistry method are composedof indicator(s) (visible colorimetric), and buffer(s).

[0035] Briefly stated, the present invention relates to test devices formeasurement of GHB or GBL in urine but could also work in otherbiological matrices such as blood, saliva, hair or other fluids and theprocedures for making said test means. This invention is in the field ofclinical diagnostics. More specifically, this invention provides drychemistry dipsticks (DCD's or on-site test modules), thin layer lateralflow chromatographic dry chemistry technology (LFD's), and thecombination of both in a unique hybrid that is not known prior to thepresent art and liquid chemistry reagents for automated and manual use.That is to say (in it's simplest terms) that this unique hybrid (LFD)will encompass the use of a dry chemistry test pad resting on thesurface of a wicking material (such as nitrocellulose) acting as a fluiddelivery device. This new art can utilize aqueous, biological specimensincluding urine, saliva, sweat extracts, blood, serum and other fluids(such as water or soft drinks, etc.). Thus, this invention provides aunique method for GHB or GBL measurement utilizing rapid test devicesincluding the automated method as well as the DCD, and LFD methodologythereby enabling in-home, workplace, and recreational testing throughover-the-counter (OTC) sales. This is an enormous advancement in theart. These advances and improvements of the present device over theprior art provides the public safety, health care and drug testingindustry with powerful new clinical and diagnostic tools.

[0036] A thorough search of the literature reveals no relative artresembling this technology; therefore, this invention is clearly a novelcreation, and is not obvious to anyone skilled in the art of toxicologyand clinical chemistry.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The instant invention is a single assay in the form of a liquidchemistry reagent, dry chemistry dipstick or lateral flow device inconjunction with using a dry chemistry test pad for the detection of GHBor GBL in sample matrices consisting of urine and other biologicalspecimens (e.g. saliva, serum, blood, sweat extracts, and hairhomogenates) or fluids such as beverages such water, soft drinks, beer,or mixed drinks. The GHB or GBL detection assay that makes up theinstant invention may take the form of dry chemistry dipsticks or drychemistry test pad lateral flow hybrid, both of which are composed ofsome or all of the following compounds: buffer(s) and colorindicator(s), hereinafter referred to as the adulteration reagent or theliquid automated reagent designed for high speed automated analyzersalso composed of some or all of the following compounds: buffer(s) andcolor indicator(s). It can be noted that the liquid reagent method couldalso be used manually employing spectrophotometers or other types ofvisual detection technology. Buffering of the reactants is critical tothe GHB or GBL reagent, because pH plays a vital role in the reactionkinetics. In the case of the dipstick (or dry chemistry dipstick, DCD)and the dipstick/lateral flow hybrid (which can be known as the “DLFH”device), GHB or GBL reagent components are impregnated on the test strippad composed of solid, absorbent carrier(s), usually known as test pads.In the case of dipsticks, these test pads are typically affixed to asolid support (usually plastic). This device is then submerged in theliquid test sample, removed, and a measurable (i.e. visible) response isobserved. Or in the case of the DLFH, the dry chemistry test pad ischemically impregnated identically to the dipstick. The test pad is thenplaced in fluid (direct) contact with lateral flow paper (such asnitrocellulose). This device is then exposed to a fluid (urine forexample). The urine then migrates to the location of the test pad,saturates the test pad, and the reaction takes place.

[0038] The GHB or GBL reagents of the device constitute the heart of theanalytical response provided by it, and is comprised of one or morereagent compositions responsive to any number of chemical componentsmade up of GHB or GBL or are analogs or precursors of GHB. The reagents,in the broadest sense produces a detectable manifestation of thepresence of GHB or GBL; the detectable manifestation can be a measurableresponse in the form of the appearance or disappearance of a color, orthe changing of one color to another. Said measurable response may alsobe evidenced by a change in the amount of light reflected or absorbedduring the reaction of interest. The analytical arts are replete withexamples of these types of detectable responses.

[0039] In the present invention, there is provided a dry chemistry teststrip in the form of a dipstick or DLFH for the detection of GHB or GBLin urine (or other biological fluids including saliva, sweat extracts,serum, blood, and hair homogenates) and fluids such as beverages toincluded but not limited to water, soft drinks, beer, or mixed drinks(possibly containing alcohol) comprising a solid, carrier matrix in theform of a dry chemistry dipstick containing an indicator compound andbuffer.

[0040] The present technology does not predict or forecast the obviousadvancement in the art to encompass the present invention, nor does ithint at the extraordinary improvement the present invention provides inthe field of GHB or GBL detection. While urine is the sample matrix ofchoice for this instant invention (and for the immunoassays currently ingeneral use for drug abuse screening) it is well within the scope ofthis novel invention to utilize it in the analysis of other samplematrices including saliva, sweat extracts, serum, hair homogenates,gastric contents, cerebral spinal fluid, blood and fluids such asbeverages to included but not limited to water, soft drinks, beer, ormixed drinks (possibly containing alcohol).

[0041] The remarkable discovery of the new art formula will require thepresence of an indicator(s) for GHB or GBL in urine (as well as theother matrices mentioned) that was unknown prior to this art. The newlydiscovered and suitable indicators and compounds that are reactive tothe presence of GHB or GBL are but, not limited to the following;hydroxybutyrate dehydrogenase, esterase, 3-hydroxybutyratedehydrogenase, 4-hydroxybutyrate dehydrogenase, carboxyl esterase,carboxylic-ester hydrolase, β-hydroxybutyrate dehydrogenase,[R]-3-hydroxybutanoate, NAD (nicotinamide adenine dinucleotide)⁺oxidoreductase, NAD (α-nicotinamide adenine dinucleotide) or analogs ofNAD such as 3-acetylpyridine adenine dinucloetide, 3-acetylpyridinehypoxanthine dinucleotide, β-nicotinamide adenine dinucleotide-agarose,nicotinamide 1,N⁶-ethenoadenine dinucleotide, nicotinamide guaninedinucleotide, nicotinamide hypoxanthine dinucleotide, nicotinic acidadenine dinucleotide, 3-pyridinealdehyde adenine dinucleotide,thionicotinamide adenine dinucleotide and α-hydroxybutyratedehydrogenase, NADP (α-nicotinamide adenine dinucleotide phosphate) oranalogs of NADP such as β-nicotinamide adenine dinucleotide phosphate,3-acetylpyridine adenine phosphate, β-nicotinamide adenine dinucleotide2′,3′-Cyclic monophosphate, β-nicotinamide adenine dinucleotide3′-phosphate, nicotinamide 1,N⁶-etheno adenine dinucleotide phosphate,nicotinamide hypoxanthine dinucleotide phosphate, thionicotinamideadenine dinucleotide phosphate and anti-gamma-hydroxybutyrate,gamma-butyrolactone dehydrogenase, anti-gamma-butyrolactone,alpha-nicotinamide adenine dinucleotide phosphate, beta-nicotinamideadenine dinucleotide phosphate and all analogs of the afore mentioned,and can be selected from the following group consisting of NBT (nitroblue tetrazolium), phenazine methosulfate, tetranitroblue tetrazolium,napthol AS-TR phosphate, methylene blue, Fast red, napthol-AS-MX,napthol AS-TR phosphate, thymol blue, bromcresol green, methyl red,cresol red, metanil yellow, m-cresol purple, xylenol blue, thymol blue,tropeolin OO, quinaldine red, α-dinitrophenol, methyl yellow; dimethylyellow, bromophenol blue, tetrabromophenol blue, bromochlorophenol blue,Congo red, methyl orange, p-ethoxychrysoidine hydrochloride, napthylred, alizarin sodium sulfonate, bromocresol green, α-dinitrophenol,methyl red, lacmoid, chlorophenol red, benzoyl auramine G, bromocresolpurple, bromophenol red, p-nitrophenol, bromthymol blue, phenol red,p-quinonemono(bis-4-oxyphenylmethide), neutral red, quinoline blue,α-naphtholphthalein, tropeolin OOO; α-napthol orange, ethylbis(2,4-dinitrophenyl)acetate, di-o-cresolphthalide, phenolphthalein,thymolphthalein, dimethylphenolphthalein, alizarin yellow GG; salicylyellow, alizarin yellow R, Nile blue,2,4,6-trinitrophenylmethyl-nitramine, tropeolin O, triphenylrosanilinesulfonic acid (sodium or potassium salt), indigo carmine, nitrobenzene,bromcresol green, bromcresol purple, bromchlorophenol blue, brilliantyellow, brilliant blue R, brilliant cresyl blue ALD, brilliant blue G,brlliant black BN, bromthymol blue, bromphenol red, bromphenol red,bromoxylenol blue, coomasie blue, azolitmin, litmus,pyrogallosulfonphthalein, pyrogallo red-molybdate, alcoholdehydrogenase, ABTS (2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonicacid)), 4-aminoantipyrine (4AAP), tetramethylbenzidine (TMB),o-phenylenediamine (OPD), o-dianisidine, 5-aminosalicylic acid (5AS),3,3′-diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC),4-chloro-1-napthol (4C1N), AEC (3-Amino-9-ethyl carbazole),dimethyl-2,5-dihydroperoxyhexane,Bis{4-[N-(3′-sulfo-n-propyl)-N-n-ethyl]amino-2,6-dimethylphenyl}methane(Bis-MAPS), N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS),N-Ethyl-N-(3-sulfopropyl)-3-methoxyaniline (ADPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)aniline (ALOS),N-Ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline (MAPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS),N-Ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS),N-(3-sulfopropyl)aniline (HALPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxy-aniline (DAOS),N-Ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline (DAPS),N-Ethyl-N-(3-sulfopropyl)aniline (ALPS),N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS),N-(3-sulfopropyl)-3,5-dimethoxyaniline (HDAPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAO),N,N-Bis(4-sulfobutyl)-3,5-dimethylaniline (MADB), pyrogallol,2,4-Dichlorophenol, N,N-Diethyl-m-toluidine, p-Hydroxybenzene Sulfonate,N,N-Dimethylaniline, 3,5-Dichloro-2-Hydroxybenzenesulfonate,2,4,6-tribromo-3-hydroxybenzoic acid, SodiumN-Ethyl-N-(3-Sulfopropyl)-m-Anisidine, hydroxybenzoic acid,4-hydroxybenzoic acid, N-Ethyl-N-(2-hydroxy-3-Sulfopropyl)-m-toluidine,AEC (3-Amino-9-ethyl carbazole), 2-5, dimethyl-2,5-dihydroperoxyhexane,Bis{4-[N-(3′-sulfo-n-propyl)-N-n-ethyl]amino-2,6-dimethylphenyl}methane(Bis-MAPS), N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS),N-Ethyl-N-(3-sulfopropyl)-3-methoxyaniline (ADPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)aniline (ALOS),N-Ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline (MAPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS),N-Ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS), N-(3-sulfopropyl)aniline (HALPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxy-aniline (DAOS),N-Ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline (DAPS),N-Ethyl-N-(3-sulfopropyl)aniline (ALPS),N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS),N-(3-sulfopropyl)-3,5-dimethoxyaniline (HDAPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAO),N,N-Bis(4-sulfobutyl)-3,5-dimethylaniline (MADB),3-Methyl-2-benzothiazolinonehydrazone, Dimethylaniline,5-bromo-6-chloro-3-indoxyl-beta-D-galacatopyranoside,4-Aminophenyl-beta-D-galactopyranoside,3-indoxyl-beta-D-galactopyranoside (blue),5-Bromo-4-chloro-3-indoxyl-beta-D-galactopyranoside (blue),5-Bromo-3-indoxyl-beta-D-galactopyranoside (blue),6-chloro-3-indoxyl-beta-D-galactopyranoside (salmon),6-Fluoro-3-indoxyl-beta-D-galactopyranoside,8-Hydroxyquinoline-beta-D-galactopyrano-side,5-Iodo-3-indoxyl-beta-D-galactopyranoside (purple),N-Methylindoxyl-beta-D-galactopyranoside,2-Nitrophenyl-beta-D-galactopyranoside,4-Nitrophenyl-beta-D-galactopyranoside, NaphtholAS-BI-beta-D-galactopyranoside, 2-Naphthyl-beta-D-galactopyranoside(yellow), 4-Methylumbelliferyl-beta-D-glucuronic acid,beta-D-Galactosidase, Iodo-3-indoxyl-beta-D-galactopyranoside,alpha-L-Galactosidase, Iodo-3-indoxyl-alpha-L-beta-Galactosidase,glycosidase, beta-Cellobiosidase, cellobioside, beta-D-Cellobiosidase,5-Bromo-4-chloro-3-indoxyl-beta-D-cellobioside,5-Bromo-6-chloro-3-indoxyl-beta-D-cellobioside,4-Nitrophenyl-beta-D-cellobioside, 1-Naphthyl-cellobioside,4-Methylumbelliferyl-beta-D-cellobioside, Arabinosidase, Fucosidase,Galactosaminidase, Glucosaminidase, Glucosidase, Glucuronidase,Lactosidase, Maltosidase, Mannosidase, and Xylosidase. Theircorresponding substrates, Arabinopyranoside, Fucopyranoside,Galactosaminide, Glucosaminide, Glucopyranoside, Glucuronic acid,Lactopyranoside, Maltopyranoside, Mannopyranoside, Xylopyranoside,5-Bromo-4-chloro-3-indoxyl, 5-Bromo-6-chloro-3-indoxyl,6-chloro-3-indoxyl, 5-Bromo-3-indoxyl, 5-Iodo-3-indoxyl, 3-indoxyl,2-(6-Bromonaphthyl), 6-Fluoro-3-indoxyl 2-Nitrophenyl, 4-Nitrophenyl,1-Naphthyl, Naphthyl AS-BI, 2-Nitrophenyl-N-acetyl,4-Nitrophenyl-N-acetyl, 4-Methylumbelliferyl, glycosidase enzyme,carboxyl esterase, cholesterol esterase, sulfatases (e.g. Arylsufatase), phosphatases (e.g. Alkaline phosphatase), carboxyl esterase,6-chloro-3-indoxyl butyrate, aryl sulfatase, 5-bromo-4-chloro-3-indoxylsulfate, alkaline phosphatase, and 2-naphthyl phosphate. It isunderstood that the present arts discovery of the use of indicators suchas the ones mentioned above or others that have not been mentioned thatare sensitive to GHB or GBL biological matrices such as urine and otherfluids are capable of producing a detectable response in the presence ofGHB or GBL are within the present art. Therefore the use of GHB or GBLindicators that are not mentioned here would fall within the spirit andscope of the present invention. These indicators form indicatorcomplexeswith GHB or GBL for determining the presence or amount ofgamma-hydroxybutyrate or gamma-butyrolactone in a sample, said method(s)comprising contacting said sample with an indicator which specificallybinds to gamma-hydroxybutyrate or gamma-butyrolactone to form anindicatorcomplex; and, measuring said indicatorcomplex to determine thepresence or amount of said gamma-hydroxybutyrate or gamma-butrylactonein said sample.

[0042] This new art formula will require appropriate buffering. Suitablebuffers may include any of the following (referred to here by theircommonly used acronyms): citrate, borate, borax, sodium tetraboratedecahydrate, sodium perchlorate, sodium chlorate, sodium carbonate, TRIS(Tris[hydroxymethyl]aminomethane), MES (2-[N-Morpholino]ethanesulfonicacid), BIS-TRIS (bis[2-Hydroxyethyl]iminotris[hydroxymethyl]methane;2-bis[2-Hydroxyethyl]amino-2-[hydroxymethyl-1,3-propanediol), ADA(N-[2-Acetamidol]-2-iminodiacetic acid; N-[Carbaoylmethyl]iminodiacetcacid), ACES (2-[(2-Amino-2-oxoethyl)amino]ethanesulfonic acid;N-[2-Acetamido]-2-aminoethanesulfonic acid), PIPES(PiperazineN-N′-bis[2-ethanesulfonic acid)]; 1,4-Piperzinedethanesulfoicacid), MOPSO (3-[N-Morpholinol]-2-hydroxypropanesulfonic acid), BIS-TRISPROPANE (1,3-bis[tris(Hydroxymethyl)methylamino]propane), BES(N,N-bis[2-Hydroxyethyl]-2-aminoethaesulfonic acid;2-bis(2-Hydroxyethyl)amino]ethanesulfonic acid), MOPS(3-[N-Morpholino]propanesulfonic acid), TES(N-tris[Hydroxymethyl]methyl-2-aminomethanesulfonic acid;2[2-Hysroxy-1,1-bis(hydroxymethyl)-ethyl]amino)ethanesulfonic acid),HEPES (N-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]), DIPSO(3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesulfonic acid), TAPSO(3-[N-tris(Hydroxyethyl)methylamino]-2-hydroxypropanesulfonic acid),HEPPSO (N-[2-Hydroxythyl]piperazine-N′-[2Hydroxypropanesulfonic acid]),POPSO (Piperazine-N,N′-bis[2-hydroxypropanesulfonic acid]), EPPS(N-[2-Hydroxyethyl]piperazine-N′-[3-propanesulfonic acid), TEA(triethanolamine), TRICINE (N-tris[Hydroxymethyl]methyllycine;N-[2-Hydroxy-1-1-bis(hydroxymethyl)etyyl]glycine), BICINE(N,N-bis[2-Hydroxyethyl]glycine), TAPS(N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid;([2-Hdroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-propanesulfonic acid),AMPSO (3-[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonicacid), CHES (2-[N-Cyclohexylamino]ethanesulfonic acid), CAPSO(3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic acid), AMP2-Amino-2-ethyl-1-propanol, CAPS (3-[cyclohexylamino]-1-propanesulfonicacid), hydrochloric acid, phosphoric acid, lactic acid, sulfuric acid,nitric acid, chromic acid, boric acid, perchloric acid, potassiumhydrogen tartrate, potassium hydrogen phthalate, calcium hydroxide,phosphate, bicarbonate, sodium hydroxide, potassium hydroxide, oxalateor succinate. Other buffers with an effective pK and pH range, andcapacity suitable for maintaining the sample-reagent mixture within therequired parameters of the assay's reaction mechanism may be added tothe above group.

[0043] Manufacture of the dry chemistry dipsticks may require theaddition of thickeners as taught in the art. Some compounds commonlyused for this purpose include: polyvinylpyrrolidone, algin, carragenin,casein, albumin, methyl cellulose, and gelatin. The typical range ofconcentration for these thickeners is about 0.5 to 5.0 g. per 100 ml.Wetting agents or surfactants are also typically used in dry chemistry.For dry chemistry applications, wetting agents aid in even distributionof the chemicals and promote even color development. Acceptable wettingagents can be hydrophilic polymers, or cationic, anionic, amphoteric, ornonionic species. Some commonly used wetting agents include sodiumdodecyl-benzene sulphonate, sodium lauryl sulphate, benzalkoniumchloride, N-lauroylsarcosine sodium salt, Brij-35, Tween 20, TritonX-100, dioctyl sodium sulphosuccinate, and polyethylene glycol 6000.Wetting agents can be added to dipstick impregnation solutions inamounts of 0.5% to 5.0%, and 0.1% to 1.0% in liquid reagents.

[0044] Color enhancers may be used such as sucrose, lactose, glucose orother compounds. Color enhancement can be defined as intensificationand/or alteration in some manner the color that is produced by thereaction to improve the measurement of the detectable response.

[0045] The production of dry chemistry test strips for the presentinvention can utilize any form of absorbent, solid phase carrierincluding filter paper, cellulose or synthetic resin fleeces inconjunction with liquid solutions of reagent compositions in volatilesolvents. This can be carried out in one or more impregnation steps.Each impregnation may contain one or more of the chemical compoundsmaking up the assay reagent composition; the exact procedure is dictatedby the inter-reactivity of the assay constituents and the order in whichthey may have to react with the analyte of interest.

[0046] In the case of the DLFH, the lateral flow invention it canutilize any form of absorbent, solid phase carrier that is capable oftransporting a fluid. These can include filter paper, cellulose orsynthetic resins. More specifically, the lateral flow material caninclude cellulose, cellulose acetate, nitrocellulose, mixed ester,teflon, polyvinylidene difluoride (PVDF), polytetrafluoroethylene(PTFE), polysulfone, cotton linter, non-woven rayon, glass fiber, nylon,ion exchange or other suitable membranes or solid support.

[0047] After impregnation, the dipsticks are dried, cut into strips,glued to a support structure (usually a flexible, flat, plastic stickmade up of polystyrene, vinyl polypropylene, and polyester or othersuitable support material) as part of a “sandwich” composed of thehandle, test pad, and a synthetic resin film and/or a fine-mesh materialin the manner described in German Pat. No, 2,118,455. In addition, theinstant invention may be combined with the water-stable film as taughtin U.S. Pat. No. 3,530,957 to produce a dipstick in which the excesssample fluid can be wiped off in order to improve the accuracy andprecision of the results.

[0048] The sensitivity of the assay can be as low as 5 ug/mL, is thecutoffs illustrated in the present art are merely illustrative. Thecutoff of 50.0 ug/mL is used because this is the range that an analgesic(high) effect is felt by the user or victim.

[0049] The following examples are provided to further illustrate theinventive aspects of the present discovery, and to further exemplifypreferred embodiments. As such, they are intended as merelyillustrative, and are not to be construed as limiting the scope of theclaims appended hereto.

EXAMPLE 1

[0050] This is a method for manufacturing a dry chemistry dipstick (DCD,test strip) with a solid carrier for the detection of GHB in urinesamples.

[0051] Filter paper is successively impregnated with the followingsolutions and dried at 25 degree C.:

[0052] Solution I

[0053] Tris-Base (Tris[hydroxymethyl]aminomethane) buffer 2.97 g

[0054] Glucose-6-phosphate 0.01M

[0055] NAD 0.3 M

[0056] distilled water added to 100 mL total volume of solution

[0057] pH the solution to a value between 1.0 and 12.5 preferably 8.0

[0058] lab notes: Buffer strength is preferably 10 mM or greater

[0059] Solution 2

[0060] GHB (gamma hydroxybutyrate) conjugated to a dehydrogenase(glucose-6-phosphate (G6P)) 5 mM

[0061] GHB antibody

[0062] Tris-HCl (Tris[hydroxymethyl]aminomethane hydrochloride) buffer0.01M

[0063] NBT (nitro blue tetrazolium) 0.01 mg/L

[0064] distilled water added to make 1000 mL total volume of solution

[0065] lab notes: pH the solution to a value between 1.0 and 12.5preferably 8.0

[0066] In this example a dipstick was prepared in accordance with theinstant invention. The device comprised a paper carrier or solid matrixincorporated with the composition of solutions 1 and 2 above. Note thatthe concentrations of any of the following examples can be varied tosuit the dipstick device format (dependent upon paper type, or use ofsemi-permeable membrane or other suitable material). This example iscarried out using the following procedure. To produce the test means, apiece of Whatman 3 MM filter paper having approximate dimensions of 1inch by 3 inches was impregnated with solution 1 by immersing the paperinto solution 1. The paper was then dried by using forced air notexceeding 25° C. A second piece of Whatman 1 MM filter paper havingapproximate dimensions of 1 inch by 3 inches was impregnated withsolution 2 by immersing the paper into solution 2. The paper was thendried by using forced air not exceeding 25° C. The dried papers are thenlaminated to each other by the use of a non-reactive (neutral adhesive).The dried, laminated papers are then applied to one side of a piece ofdouble-sided adhesive transfer tape commercially available from 3MCompany, St. Paul, Minn. 55144. The laminate is then slit into portionsmeasuring 3 inches by 0.2 inches. One portion is then attached, via theunused adhesive side to a polystyrene sheet measuring about 1.5 inchesby 3 inches and the resulting laminate is slit parallel to its shortdimension to form test devices comprising a 1.5 inch oblong polystyrenestrip carrying a square of the impregnated papers at one end, the otherend serving as the handle. When the dipstick thus obtained is dippedinto a urine (note: other sample matrices including saliva, sweatextracts, serum, hair homogenates, gastric contents, cerebral spinalfluid, blood and fluids such as beverages to included but not limited towater, soft drinks, beer, or mixed drinks) will produce no uniform bluecolor development if no GHB is present. Conversely, if any concentrationof GHB is present in the urine at a 0.1% v/v or greater a blue colorwill develop thus confirming the presence of GHB.

[0067] In summary, Example 1 is as follows: the foregoing dry chemistrytest strip (dipstick) method for the GHB detection of in a sample ofurine comprises the steps of preparing a test means by successivelyimpregnating a solid, carrier matrix with reagent solutions, drying theimpregnated, solid test means, then dipping said dried test means intourine, and finally observing any color change in the presence or absenceof GHB.

[0068] The following changes to the above reagent solutions will remainwithin the scope and function of this invention and will have similarresults to the example above. The GHB indicator reactive compound(reactive in this sense means that the GHB conjugated to thedehydrogenase competes with free GHB if present for the antibody)dehydrogenase (glucose-6-phosphate dehydrogenase (G6PDH)) is sensitivethe presence of glucose-6-phosphate in solution. If there is no free GHBin the specimen then the dehydrogenase will not react with the G6Pbecause it will be bound up by the GHB antibody that will bind to theGHB conjugated to the G6PDH, note that during the G6PDH-G6P reaction NAD(cofactor) will be converted to NADH and in the presence of NADH, NBTwill change from a colorless compound to a blue/purple color. If thereis GHB present in the sample it will be will bind with the GHB antibodyleaving free GHB-G6PDH conjugate free to react with G6P thus causing thecascade reaction of the NAD conversion to NADH and the subsequentconversion of the NBT indicator. In solution 1 the dehydrogenase(G6PDH), which could be substituted with one or more of the followingcompounds including hydroxybutyrate dehydrogenase, esterase,3-hydroxybutyrate dehydrogenase, 4-hydroxybutyrate dehydrogenase,carboxyl esterase, carboxylic-ester hydrolase, β-hydroxybutyratedehydrogenase, [R]-3-hydroxybutanoate, NAD (nicotinamide adeninedinucleotide)⁺ oxidoreductase, and α-hydroxybutyrate dehydrogenase,anti-gamma-hydroxybutyrate, alpha-nicotinamide adenine dinucleotidephosphate, beta-nicotinamide adenine dinucleotide phosphate,dehydrogenase, α-hydroxybutyrate dehydrogenase,anti-gamma-hydroxybutyrate, alpha-nicotinamide adenine dinucleotidephosphate, beta-nicotinamide adenine dinucleotide phosphate, oxidases,reductases, oxidoreductases, transferases, hydrolases, lyases,isomerases and ligases and all analogs of the afore mentioned can besubstituted with the following enzyme pairs to include but are notlimited to one or more of the following:4-Aminophenyl-beta-D-galactopyranoside,3-indoxyl-beta-D-galactopyranoside (blue),5-Bromo-4-chloro-3-indoxyl-beta-D-galactopyranoside (blue),5-Bromo-3-indoxyl-beta-D-galactopyranoside (blue),6-chloro-3-indoxyl-beta-D-galactopyranoside (salmon),6-Fluoro-3-indoxyl-beta-D-galactopyranoside,8-Hydroxyquinoline-beta-D-galactopyranoside,5-Iodo-3-indoxyl-beta-D-galactopyranoside (purple),N-Methylindoxyl-beta-D-galactopyranoside,2-Nitrophenyl-beta-D-galactopyranoside,4-Nitrophenyl-beta-D-galactopyranoside, NaphtholAS-BI-beta-D-galactopyranoside, and 2-Naphthyl-beta-D-galactopyranoside(yellow). Fluorescent substrates may also be utilized including4-Methylumbelliferyl-beta-D-glucuronic acid. The colors noted in theparentheses are those produced in the reaction described above. Theindicator substrate used in these examples must be matched to theconformation of the galactosidase used (i.e. alpha or beta, anddextrorotorary (D) or levorotorary (L)). For example,beta-D-Galactosidase should be matched with the indicator/substrateIodo-3-indoxyl-beta-D-galactopyranoside; conversely,alpha-L-Galactosidase would be matched withIodo-3-indoxyl-alpha-L-galactopyranoside. Note that somecross-reactivity does occur between stereo-isomers and, therefore, it ispossible to substitute these compounds where appropriate.

[0069] Substitution of the G6PDH with another enzyme would necessitate achange of substrate indicator complex. If another glycosidase wasselected, it would have to be matched to the appropriate substrate (e.g.beta-Cellobiosidase and a cellobioside). Examples of substrates forbeta-D-Cellobiosidase include5-Bromo-4-chloro-3-indoxyl-beta-D-cellobioside,5-Bromo-6-chloro-3-indoxyl-beta-D-cellobioside,4-Nitrophenyl-beta-D-cellobioside, 1-Naphthyl-cellobioside,beta-Galactosidase and the fluorescent indicator,4-Methylumbelliferyl-beta-D-cellobioside.

[0070] Other glycosidases which may be substituted for Galactosidase andCellobiosidase include the alpha and beta, and D and L conformations ofthe following enzymes: Arabinosidase, Fucosidase, Galactosaminidase,Glucosaminidase, Glucosidase, Glucuronidase, Lactosidase, Maltosidase,Mannosidase, and Xylosidase. Their corresponding substrates,Arabinopyranoside, Fucopyranoside, Galactosaminide, Glucosaminide,Glucopyranoside, Glucuronic acid, Lactopyranoside, Maltopyranoside,Mannopyranoside, and Xylopyranoside may be bound to each of thefollowing color indicator groups: 5-Bromo-4-chloro-3-indoxyl,5-Bromo-6-chloro-3-indoxyl, 6-chloro-3-indoxyl, 5-Bromo-3-indoxyl,5-Iodo-3-indoxyl, 3-indoxyl, 2-(6-Bromonaphthyl), 6-Fluoro-3-indoxyl2-Nitrophenyl, 4-Nitrophenyl, 1-Naphthyl, Naphthyl AS-BI,2-Nitrophenyl-N-acetyl, 4-Nitrophenyl-N-acetyl, and 4-Methylumbelliferylmoieties.

[0071] Other enzymes that can be used with the indicator groups listedabove. These include esterases (e.g. Carboxyl esterase, and Cholesterolesterase), sulfatases (e.g. Aryl sufatase), and phosphatases (e.g.Alkaline phosphatase). These enzymes can utilize the indicator groupsdelineated above when conjugated to the corresponding substrate. Forexample, Carboxyl esterase and 6-chloro-3-indoxyl butyrate, and Arylsulfatase and 5-bromo-4-chloro-3-indoxyl sulfate, and Alkalinephosphatase and 2-naphthyl phosphate form enzyme-substrate pairs.

[0072] Other enzymes may be conjugated to GHB, and therefore substitutedfor the species described above. This group now listed, however, mustutilize a substrate that is distinct and separate from the indicator.This enzyme group may include any dehydrogenase, oxidase, hydroxylase,or oxidoreductase. Each grouping will utilize a specific indicator orgroup of indicators. The dehydrogenases and hydroxylases will utilize aco-enzyme, a color indicator and an electron carrier such as a-NAD(a-Nicotinamide adenine dinucleotide), however this electroncarrier/acceptor can be replaced by the alpha or beta isomers of any oneof the following substitutes: nicotinamide adenine dinucleotide,nicotinamide adenine dinucleotide 3′-phosphate, nicotinamide adeninedinucleotide phosphate, triphosphopyridine, nicotinamide1-N1-ethenoadenine dinucleotide phosphate, nicotinamide hypoxanthinedinucleotide, nicotinamide hypoxanthine dinucleotide phosphate,nicotinamide mononucleotide, nicotinamide N1-propylsulfonate,nicotinamide ribose monophosphate, or other analogs of NAD.

[0073] Some dehydrogenases and hydroxylases and their substrate pairswhich can be used include Formaldehyde dehydrogenase and Formaldehyde,Fructose dehydrogenase and Fructose, Glucose-6-phosphate dehydrogenaseand Glucose-6-phosphate, Glucose dehydrogenase and Glucose, Glutamatedehydrogenase and Glutamate, Glycerol dehydrogenase and Glycerol,Glycerol-3-phosphate dehydrogenase and Glycerol-3-phosphate,Hydroxybutyrate dehydrogenase and Hydroxybutyrate, Hydroxybenzoatehydroxylase and 4-Hydroxybenzoate, Lactate dehydrogenase and Lactate,Leucine dehydrogenase and Leucine, Malate dehydrogenase and Malate,Mannitol dehydrogenase and Mannitol, or any other dehydrogenase orhydroxylase.

[0074] The use of oxidases to replace the gylcosidase also requires aseparate indicator, and peroxidase. Some oxidases and their substratepair which can be used include Acyl-CoA oxidase and Acyl-CoA, Alcoholoxidase and Ethanol, Ascorbate oxidase and Ascorbate, Cholesteroloxidase and Cholesterol, Choline oxidase and Choline, Glucose oxidaseand Glucose, Glycerophosphate oxidase and Glycerophosphate, Xanthineoxidase and Xanthine, Uricase and Uric acid, or any other oxidase or allanalogs of the afore mentioned.

[0075] The indicator NBT, which will develop color in the presence ofthe reduction of NAD or NADPH can be replaced by one of the following:phenazine methosulfate, tetranitroblue tetrazolium, napthol AS-TRphosphate, methylene blue, Fast red, napthol-AS-MX, napthol AS-TRphosphate or analogs thereof.

[0076] The Tris buffer in solution 1, may be substituted with one ormore of the following buffers: citrate, borate, borax, sodiumtetraborate decahydrate, sodium perchlorate, sodium chlorate, sodiumcarbonate, MES (2-[N-Morpholino]ethanesulfonic acid), BIS-TRIS(bis[2-Hydroxyethyl]iminotris[hydroxymethyl]methane;2-bis[2-Hydroxyethyl]amino-2-[hydroxymethyl-1,3-propanediol), ADA(N-[2-Acetamidol]-2-iminodiacetic acid; N-[Carbaoylmethyl]iminodiacetcacid), ACES (2-[(2-Amino-2-oxoethyl)amino]ethanesulfonic acid;N-[2-Acetamido]-2-aminoethanesulfonic acid), PIPES(PiperazineN-N′-bis[2-ethanesulfonic acid)]; 1,4-Piperzinedethanesulfoicacid), MOPSO (3-[N-Morpholinol]-2-hydroxypropanesulfonic acid), BIS-TRISPROPANE (1,3-bis[tris(Hydroxymethyl)methylamino]propane), BES(N,N-bis[2-Hydroxyethyl]-2-aminoethaesulfonic acid;2-bis(2-Hydroxyethyl)amino]ethanesulfonic acid), MOPS(3-[N-Morpholino]propanesulfonic acid), TES(N-tris[Hydroxymethyl]methyl-2-aminomethanesulfonic acid;2[2-Hysroxy-1,1-bis(hydroxymethyl)-ethyl]amino)ethanesulfonic acid),HEPES (N-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]), DIPSO(3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesulfonic acid), TAPSO(3-[N-tris(Hydroxyethyl)methylamino]-2-hydroxypropanesulfonic acid),HEPPSO (N-[2-Hydroxythyl]piperazine-N′-[2Hydroxypropanesulfonic acid]),POPSO (Piperazine-N,N′-bis[2-hydroxypropanesulfonic acid]), EPPS(N-[2-Hydroxyethyl]piperazine-N′-[3-propanesulfonic acid), TEA(triethanolamine), TRICINE (N-tris[Hydroxymethyl]methyllycine;N-[2-Hydroxy-1-1-bis(hydroxymethyl)etyyl]glycine), BICINE(N,N-bis[2-Hydroxyethyl]glycine), TAPS(N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid;([2-Hdroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-propanesulfonic acid),AMPSO (3-[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonicacid), CHES (2-[N-Cyclohexylamino]ethanesulfonic acid), CAPSO(3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic acid), AMP2-Amino-2-ethyl-1-propanol, CAPS (3-[cyclohexylamino]-1-propanesulfonicacid), hydrochloric acid, phosphoric acid, lactic acid, sulfuric acid,nitric acid, chromic acid, boric acid, perchloric acid, potassiumhydrogen tartrate, potassium hydrogen phthalate, calcium hydroxide,phosphate, bicarbonate, sodium hydroxide, potassium hydroxide, oxalateor succinate. Other buffers with an effective pK and pH range, andcapacity suitable for maintaining the sample-reagent mixture within therequired parameters of the assay's reaction mechanism may be added tothe above group.

EXAMPLE 2

[0077] This is a method for manufacturing a liquid, carrier-free reagentfor the adulteration detection of GHB in samples submitted for drugs ofabuse analysis.

[0078] Prepare a Solution Containing:

[0079] Solution I (R1)

[0080] Tris-Base (Tris[hydroxymethyl]aminomethane) buffer 2.97 g

[0081] Iodo-3-indoxyl-beta-D-galactopyranoside 0.01M

[0082] distilled water added to 1000 mL total volume of solution

[0083] pH the solution to a value between 1.0 and 12.5 preferably 8.0

[0084] lab notes: Buffer strength is preferably 10 mM or greater

[0085] Solution II (R2)

[0086] GHB (gamma hydroxybutyrate) conjugated to galactosidase 5 mM

[0087] GHB antibody

[0088] Tris-HCl (Tris[hydroxymethyl]aminomethane hydrochloride) buffer0.01M

[0089] distilled water added to make 1000 mL total volume of solution

[0090] lab notes: pH the solution to a value between 1.0 and 12.5preferably 8.0

[0091] In this example a dipstick was prepared in accordance with theinstant invention.

[0092] GHB Calibrator Formulations

[0093] Zero (0) Calibrator:

[0094] 1 liter of 0.2 micron filtered normal human urine with noadulterants or drugs present, and 0.01% sodium azide.*

[0095] 50 ug/mL Calibrator:

[0096] 50 ug/mL GHB

[0097] 0.01 M Sodium Borate

[0098] 100 mL of 0.2 micron filtered normal human urine with noadulterants or drugs present*

[0099] pH the solution to a value between 3.0 and 11.0 preferably 9.0.

[0100] lab notes:* Human urine can be substituted with distilled water,synthetic urine or other suitable solvent. The bacterial inhibitorsodium azide could be replaced with chloroamphenicol or other suitablebacterial inhibitors that would inhibit the growth of bacteria.

[0101] The reagent system of the instant invention (liquid reagent) isintended for use on any automatic chemistry analyzers with open channelcapability including Olympus series, Hitachi 700 series, Beckmans andmany others. The reagent as outlined in Example 2 is used in thefollowing manner: the one component of the reagent composition (R-1) isplaced in the reagent compartment of the analyzer; samples, calibrators,and controls are aliquoted into sample cups, which are then placed onthe analyzer. An aliquot of 5 uL of each specimen is then pipetted intoa single, discrete cuvette followed by the addition of 150 uL of thefirst reagent, R-1, and mixed; A first spectrophotometer reading is thentaken followed by a second after a specified incubation period (i.e. oneminute for this example) at the specified wavelength (between 340 and800 nm). The spectrophotometer readings are then recorded. In thisinstance the assay is read at 415 nm. The absorbance of samples, andcontrols are printed and then compared to the calibrator's absorbance.The quantitative value for GHB concentration is then calculated. Anyconcentration of GHB greater than 50.0 ug/mL is considered positive forthe presence of GHB.

[0102] Please note if the present art is not used as illustrated thatvery significant increase in the cost of analysis, because a GC-MS assaymust then be performed to verify the presence of GHB. The GC-MS analysiscosts 100 times as much as the screen ($100 vs $1). Every additionalunnecessary GC-MS performed drives up the overall cost of drug testing.Eliminating these additional, unnecessary assays will save millions ofdollars per year.

[0103] Specifications for running urine samples vary from instrument toinstrument. Listed below is an example of parameters for the Hitachi 700series analyzer. The settings are intended as guidelines, and are setforth with the understanding that all those skilled in the art wouldrecognize that such parameters will vary from instrument to instrument.

[0104] The suggested specifications for the Hitachi 700 series are asfollows:

[0105] Parameter Settings for the Hitachi 700 Series Test: [GHB] Assaycode: [1 POINT] [50]-[0] Sample volume: [5] [5] R1 volume [125] [100][NO] R2 volume [125] [100] [NO] Wavelength [0] [415] Calib. Method:[Linear] [0] [0] Std. (1) Conc.-POS: [0.0]*-[1]* assigned calibratorvalue Std. (2) Conc.-POS: [50.0]-[2 ] assigned calibrator value Std. (3)Conc.-POS: [ ]-[ ] Std. (4) Conc.-POS: [ ]-[ ] Std. (5) Conc.-POS: [ ]-[] Std. (6) Conc.-POS: [ ]-[ ] SD Limit: [999] Duplicate Limit: [32000]Sensitivity Limit: [0] ABS. Limit (INC/DEC): [32000] [INCREASE] ProzoneLimit: [0] [lower] Expected Value: [0.0]-[1.0] Tech. Limit: [0]-[1000]Instrument Factor [1.0]

[0106] Thus as described above, an unknown urine submitted for drugs ofanaylsis for GHB will produce a value of less than the 0.0 ug/mL if noGHB is present. Conversely, if the sample has a concentration of greaterthan 50.0 ug/mL than the sample is positive for GHB.

[0107] To summarize more specifically Example 2, the automated methodfor the detection of adulteration of an unknown sample of urinesubmitted for drugs of abuse testing comprising the steps of placingaliquots of an unknown urine (or other biological sample i.e. serum,whole blood, cerebral spinal fluid, gastric fluid, hair homogenates,sweat extracts, saliva or other biological fluid and other fluids suchas beverages, water, etc.) and calibrator to be tested in automatedanalyzer sampling cups, placing the cups in a sampling tray within anautomated analyzer, transferring the aliquots of sample and calibratorto cuvettes mounted within the automated analyzer, injecting a firstreagent composition (R-1) comprising an indicator and buffer in anaqueous medium into the cuvettes, and mixing sample and reagents, andreading absorbance values of reaction mixture composed of reagents andtest samples (said test samples include urine specimens, controls, andcalibrator) at specified intervals, in accordance with a preprogrammedcode introduced into the automated analyzer, at a preprogrammedmonochromatically specified wavelength, and comparing absorbance of thefirst reagent composition plus the unknown samples with that of thefirst reagent composition plus the calibrator containing a zeroreference point (normal urinary matrix), and thereby determiningquantitatively the presence or absence of GHB.

[0108] The following changes to the above reagent solutions will remainwithin the scope and function of this invention and will have similarresults to the example above. The GHB indicator reactive compound(reactive in this sense means that the GHB conjugated to thegalactosidase competes with free GHB if present for the antibody)galactosidase is sensitive the presence ofiodo-3-indoxyl-beta-D-galactopyranoside in solution. If there is no freeGHB in the specimen then the galactosidase will not react with theiodo-3-indoxyl-beta-D-galactopyranoside because it will be bound up bythe GHB antibody that will bind to the GHB conjugated to thegalactosidase. If there is GHB present in the sample it will be willbind with the GHB antibody leaving free GHB-galactosidase conjugate freeto react with iodo-3-indoxyl-beta-D-galactopyranoside thus causing thedevelopment of color which can be monitored by the spectrophotometer atthe specified wavelength. In solution 1 the galactosidase, which couldbe substituted with one or more of the following compounds includinghydroxybutyrate dehydrogenase, esterase, 3-hydroxybutyratedehydrogenase, 4-hydroxybutyrate dehydrogenase, carboxyl esterase,carboxylic-ester hydrolase, β-hydroxybutyrate dehydrogenase,[R]-3-hydroxybutanoate, NAD (nicotinamide adenine dinucleotide)⁺oxidoreductase, and α-hydroxybutyrate dehydrogenase,anti-gamma-hydroxybutyrate, alpha-nicotinamide adenine dinucleotidephosphate, beta-nicotinamide adenine dinucleotide phosphate,dehydrogenase, oxidases, reductases, oxidoreductases, transferases,hydrolases, lyases, isomerases, glycosidases, phosphatases, sulfatases,ligases and all analogs of the afore mentioned can be substituted withthe following can be substituted with the following enzyme pairs toinclude but are not limited to one or more of the following:4-Aminophenyl-beta-D-galactopyranoside,3-indoxyl-beta-D-galactopyranoside (blue),5-Bromo-4-chloro-3-indoxyl-beta-D-galactopyranoside (blue),5-Bromo-3-indoxyl-beta-D-galactopyranoside (blue),6-chloro-3-indoxyl-beta-D-galactopyranoside (salmon),6-Fluoro-3-indoxyl-beta-D-galactopyranoside,8-Hydroxyquinoline-beta-D-galactopyrano-side,5-Iodo-3-indoxyl-beta-D-galactopyranoside (purple),N-Methylindoxyl-beta-D-galactopyranoside,2-Nitrophenyl-beta-D-galactopyranoside,4-Nitrophenyl-beta-D-galactopyranoside, NaphtholAS-BI-beta-D-galactopyranoside, and 2-Naphthyl-beta-D-galactopyranoside(yellow). Fluorescent substrates may also be utilized including4-Methylumbelliferyl-beta-D-glucuronic acid. The colors noted in theparentheses are those produced in the reaction described above. Theindicator substrate used in these examples must be matched to theconformation of the galactosidase used (i.e. alpha or beta, anddextrorotorary (D) or levorotorary (L)). For example,beta-D-Galactosidase should be matched with the indicator/substrateIodo-3-indoxyl-beta-D-galactopyranoside; conversely,alpha-L-Galactosidase would be matched withIodo-3-indoxyl-alpha-L-galactopyranoside. Note that somecross-reactivity does occur between stereo-isomers and, therefore, it ispossible to substitute these compounds where appropriate.

[0109] Substitution of the glactosidase with another enzyme wouldnecessitate a change of substrate indicator complex. If anotherglycosidase was selected, it would have to be matched to the appropriatesubstrate (e.g. beta-Cellobiosidase and a cellobioside). Examples ofsubstrates for beta-D-Cellobiosidase include5-Bromo-4-chloro-3-indoxyl-beta-D-cellobioside,5-Bromo-6-chloro-3-indoxyl-beta-D-cellobioside,4-Nitrophenyl-beta-D-cellobioside, 1-Naphthyl-cellobioside,beta-Galactosidase and the fluorescent indicator,4-Methylumbelliferyl-beta-D-cellobioside.

[0110] Other glycosidases which may be substituted for Galactosidase andCellobiosidase include the alpha and beta, and D and L conformations ofthe following enzymes: Arabinosidase, Fucosidase, Galactosaminidase,Glucosaminidase, Glucosidase, Glucuronidase, Lactosidase, Maltosidase,Mannosidase, and Xylosidase. Their corresponding substrates,Arabinopyranoside, Fucopyranoside, Galactosaminide, Glucosaminide,Glucopyranoside, Glucuronic acid, Lactopyranoside, Maltopyranoside,Mannopyranoside, and Xylopyranoside may be bound to each of thefollowing color indicator groups: 5-Bromo-4-chloro-3-indoxyl,5-Bromo-6-chloro-3-indoxyl, 6-chloro-3-indoxyl, 5-Bromo-3-indoxyl,5-Iodo-3-indoxyl, 3-indoxyl, 2-(6-Bromonaphthyl), 6-Fluoro-3-indoxyl2-Nitrophenyl, 4-Nitrophenyl, 1-Naphthyl, Naphthyl AS-BI,2-Nitrophenyl-N-acetyl, 4-Nitrophenyl-N-acetyl, and 4-Methylumbelliferylmoieties.

[0111] Other enzymes that can be used with the indicator groups listedabove. These include esterases (e.g. Carboxyl esterase, and Cholesterolesterase), sulfatases (e.g. Aryl sufatase), and phosphatases (e.g.Alkaline phosphatase). These enzymes can utilize the indicator groupsdelineated above when conjugated to the corresponding substrate. Forexample, Carboxyl esterase and 6-chloro-3-indoxyl butyrate, and Arylsulfatase and 5-bromo-4-chloro-3-indoxyl sulfate, and Alkalinephosphatase and 2-naphthyl phosphate form enzyme-substrate pairs.

[0112] Other enzymes may be conjugated to GHB, and therefore substitutedfor the species described above. This group now listed, however, mustutilize a substrate that is distinct and separate from the indicator.This enzyme group may include any dehydrogenase, oxidase, hydroxylase,or oxidoreductase. Each grouping will utilize a specific indicator orgroup of indicators. The dehydrogenases and hydroxylases will utilize aco-enzyme, a color indicator and an electron carrier such as a-NAD(a-Nicotinamide adenine dinucleotide), however this electroncarrier/acceptor can be replaced by the alpha or beta isomers of any oneof the following substitutes: nicotinamide adenine dinucleotide,nicotinamide adenine dinucleotide 3′-phosphate, nicotinamide adeninedinucleotide phosphate, triphosphopyridine, nicotinamide1-N1-ethenoadenine dinucleotide phosphate, nicotinamide hypoxanthinedinucleotide, nicotinamide hypoxanthine dinucleotide phosphate,nicotinamide mononucleotide, nicotinamide N1-propylsulfonate,nicotinamide ribose monophosphate, or other analogs of NAD.

[0113] Some dehydrogenases and hydroxylases and their substrate pairswhich can be used include Formaldehyde dehydrogenase and Formaldehyde,Fructose dehydrogenase and Fructose, Glucose-6-phosphate dehydrogenaseand Glucose-6-phosphate, Glucose dehydrogenase and Glucose, Glutamatedehydrogenase and Glutamate, Glycerol dehydrogenase and Glycerol,Glycerol-3-phosphate dehydrogenase and Glycerol-3-phosphate,Hydroxybutyrate dehydrogenase and Hydroxybutyrate, Hydroxybenzoatehydroxylase and 4-Hydroxybenzoate, Lactate dehydrogenase and Lactate,Leucine dehydrogenase and Leucine, Malate dehydrogenase and Malate,Mannitol dehydrogenase and Mannitol, or any other dehydrogenase orhydroxylase.

[0114] The use of oxidases to replace the glycosidase also requires aseparate indicator, and peroxidase. Some oxidases and their substratepair which can be used include Acyl-CoA oxidase and Acyl-CoA, Alcoholoxidase and Ethanol, Ascorbate oxidase and Ascorbate, Cholesteroloxidase and Cholesterol, Choline oxidase and Choline, Glucose oxidaseand Glucose, Glycerophosphate oxidase and Glycerophosphate, Xanthineoxidase and Xanthine, Uricase and Uric acid, or any other oxidase or allanalogs of the afore mentioned.

[0115] The Tris buffer in solution 1, may be substituted with one ormore of the following buffers: citrate, borate, borax, sodiumtetraborate decahydrate, sodium perchlorate, sodium chlorate, sodiumcarbonate, MES (2-[N-Morpholino]ethanesulfonic acid), BIS-TRIS(bis[2-Hydroxyethyl]iminotris[hydroxymethyl]methane;2-bis[2-Hydroxyethyl]amino-2-[hydroxymethyl-1,3-propanediol), ADA(N-[2-Acetamidol]-2-iminodiacetic acid; N-[Carbaoylmethyl]iminodiacetcacid), ACES (2-[(2-Amino-2-oxoethyl)amino]ethanesulfonic acid;N-[2-Acetamido]-2-aminoethanesulfonic acid), PIPES(PiperazineN-N′-bis[2-ethanesulfonic acid)]; 1,4-Piperzinedethanesulfoicacid), MOPSO (3-[N-Morpholinol]-2-hydroxypropanesulfonic acid), BIS-TRISPROPANE (1,3-bis[tris(Hydroxymethyl)methylamino]propane), BES(N,N-bis[2-Hydroxyethyl]-2-aminoethaesulfonic acid;2-bis(2-Hydroxyethyl)amino]ethanesulfonic acid), MOPS(3-[N-Morpholino]propanesulfonic acid), TES(N-tris[Hydroxymethyl]methyl-2-aminomethanesulfonic acid; 2[2-Hysroxy-1,1-bis(hydroxymethyl)-ethyl]amino)ethanesulfonic acid), HEPES(N-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]), DIPSO(3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesulfonic acid), TAPSO(3-[N-tris(Hydroxyethyl)methylamino]-2-hydroxypropanesulfonic acid),HEPPSO (N-[2-Hydroxythyl]piperazine-N′-[2Hydroxypropanesulfonic acid]),POPSO (Piperazine-N,N′-bis[2-hydroxypropanesulfonic acid]), EPPS(N-[2-Hydroxyethyl]piperazine-N′-[3-propanesulfonic acid), TEA(triethanolamine), TRICINE (N-tris[Hydroxymethyl]methyllycine;N-[2-Hydroxy-1-1-bis(hydroxymethyl)etyyl]glycine), BICINE(N,N-bis[2-Hydroxyethyl]glycine), TAPS(N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid;([2-Hdroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-propanesulfonic acid),AMPSO (3-[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonicacid), CHES (2-[N-Cyclohexylamino]ethanesulfonic acid), CAPSO(3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic acid), AMP2-Amino-2-ethyl-1-propanol, CAPS (3-[cyclohexylamino]-1-propanesulfonicacid), hydrochloric acid, phosphoric acid, lactic acid, sulfuric acid,nitric acid, chromic acid, boric acid, perchloric acid, potassiumhydrogen tartrate, potassium hydrogen phthalate, calcium hydroxide,phosphate, bicarbonate, sodium hydroxide, potassium hydroxide, oxalateor succinate. Other buffers with an effective pK and pH range, andcapacity suitable for maintaining the sample-reagent mixture within therequired parameters of the assay's reaction mechanism may be added tothe above group.

EXAMPLE 3

[0116] This example will illustrate in detail the exact method formanufacturing the lateral flow GHB method. Keep in mind this methodcould be utilized for any general chemistry “test pad” or pads that arecurrently used or will be used in the art. In the case of DLFHtechnology, the manufacturing process includes impregnating onto anabsorbent, solid carrier (e.g. paper) called in this example, the “testpad”, in exactly the same manner as Example 1 with similar constituents.The test pad, once impregnated, is dried, then mounted onto a solidsupport (nitrocellulose membrane) that is capable of transporting(through lateral flow) liquid to the test pad from the point ofapplication of a test sample. In simpler terms, the device is dippedinto a liquid or the liquid sample is placed on the device at the bottomor starting point for the assay. The liquid migrates from the startingapplication point to the opposite end of the nitrocellulose lateral flowpaper, during which the test pad becomes saturated with the sample. Thereaction takes place on the test pad and color develops. The developedcolor is then compared to a color chart with known concentrations of GHBthat has the appropriate colors relative to each specific concentrationof GHB(s). For example a specific color for 0.0 ug/mL GHB, 50.0 ug/mL,100.0 ug/mL, etc., for comparison. The results are then recorded. Note,the test pad must be an absorbent (wicking) material that permitsmigration of sample up the solid absorbent test pad and allows analytesand reactants to interact.

[0117] Absorbent material is successively impregnated with the followingsolutions and dried at 25 degree C.:

[0118] Solution 1

[0119] NAD 0.3 M

[0120] hydroxybutyrate dehydrogenase 5 mM

[0121] Tris-HCl (Tris[hydroxymethyl]aminomethane hydrochloride) buffer0.01M

[0122] NBT (nitro blue tetrazolium) 0.01 mg/L

[0123] distilled water added to 100 mL total volume of solution

[0124] pH the solution to a value between 1.0 and 12.5 preferably 8.0

[0125] lab notes: Buffer strength is preferably 10 mM or greater

[0126] In this example, the lateral flow device is prepared inaccordance with the instant invention. The lateral flow device iscomprised of a paper carrier matrix (S&S, 593 grade filter paper)impregnated with the compositions of solution 1. The paper is then cutinto test pads 5 mm by 5 mm. Note that said concentrations of any of theabove constituents can be varied to suit the DLFH lateral flow/dipstickdevice format (e.g. dependent upon paper type, and inclusion ofsemi-permeable membranes or other innovations utilized in dry chemistrytechnology). The paper is then dried using forced air. The driedimpregnated test pad is then placed at approximately 35 mm (in themiddle) of a 5 mm wide by 70 mm long nitrocellulose membrane (S&SFastTrack™ NC) and makes fluid contact with nitrocellulose lateral flowpaper. The nitrocellulose membrane is capable of transporting a liquidby capillary action or wicking from one end of the lateral flow deviceto the other in approximately 60 seconds. In this example, the DLFH hasthe dimensions of 5 mm wide by 70 mm long and can be backed by or incontact with strips of glass fiber filter material (e.g. S&S 30 grade)to aid in controlling the wicking action, or other solid supportmaterial can be used.

[0127] Again, to completely illustrate the present device the startingpoint or origin at which the sample is placed on the test device is 5 mmfrom one end of the strip, and 35 mm from the site of where the test padis placed in fluid contact with the strip. For simplicity, this examplewill have the 5 mm by 5 mm impregnated test pad placed on top of thelateral flow paper and thus be in fluid contact with the said paper.

[0128] The mechanics of how the present art's LFD and dipstick test padhybrid may be explained is as follows. The starting point or origin atwhich the sample is placed on the test device is 5 mm from one end ofthe strip, and 35 mm from site where the chemically impregnated test padis in fluid contact with the lateral flow paper. The test pad can beplaced on top of the lateral flow paper making fluid contact with thelateral flow paper from the bottom side of the test pad, or the lateralflow paper can touch the paper from the side of the test pad and remainin fluid contact with the test pad. Or the lateral flow paper can reston top of the edge of test pad or be attached and in fluid contact withthe test pad in some other manner. One of the novel advantages in usinga hybrid device made of lateral flow material and a dry chemistry testpad is the lack of cross contamination from one pad to the next fromexcessive fluid, as is inherent in the prior art. For illustration,currently there are available many different types of dry chemistry teststrips available, such as the Miles Laboratories, Inc. MULTISTIXref.This device and many other like it has multiple reagents test pads withdifferent chemistries impregnated onto each pad on a single supportmembrane backing (usually plastic). Because of the relative proximity ofthese pads to each other on the same device it is easy for crosscontamination to occur, causing unreliable results. This is called“runover” (i.e. when a reagent from one pad runs over another adjacenttest pad). The present arts eliminates runover. The applicant's novelapproach to the solution of runover has not been taught prior to thepresent art and is the result of extensive research and development.

[0129] Result interpretation can be explained as follows. If the sampleis positive, with a concentration of 50.0 ug/mL GHB or more, thefollowing occurs. A drop of urine (approximately 50 uL) is applied atthe starting point or origin of the strip. The urine then migrates tothe opposite or terminal end of the strip. As the urine migrates acrossthe lateral flow material (nitrocellulose) and comes into contact withthe test pad (filter paper), the urine will saturate the pad and cause achemical reaction between the impregnated chemicals and GHB in theurine. A blue color will develop on the test pad indicating a positive(greater than 50.0 ug/mL GHB) for the presence of high levels of GHB.This color can then be compared to a color chart showing the differentcolors from colorless (white background)) to a dark blue depending uponthe concentration of the GHB(s), if greater than 50.0 ug/mL. Thereaction on the test pad is immediate thus the test results can beobserved immediately.

[0130] If the sample is negative, with a concentration of less than 50.0ug/mL of GHB present the following occurs. A drop of urine(approximately 50 uL) is applied at the starting point or origin of thestrip. The urine then migrates to the opposite or terminal end of thestrip. As the urine migrates across the lateral flow material and comesinto contact with the test pad, the urine will saturate the pad andcause a chemical reaction between the impregnated chemicals and GHB.However, this example is for a negative result, thus, no reaction occursand no color develops, indicating a negative result. This negativeresult color can then be compared to a color chart showing the differentcolors from no color developed (negative) to dark blue depending uponthe concentration of the GHB, if greater than 50 ug/mL GHB. The reactionon the test pad is immediate thus the test results can be observedimmediately.

[0131] Changes to the above reagent solution of example 3 can be madeand still remain within the scope and function of this invention andwill have similar results to examples 1 and 2 above. The indicator(s)and buffer(s) of example 3 can be replaced by all the examples andpossible substitutions as illustrated in example 1.

[0132] This brief description of the present art illustrates acompletely enabled device that would allow an individual, physician,patient, and/or technician to quickly and easily determine the presenceof the GHB in urine, providing a much needed advancement the art of GHBtesting.

[0133] To briefly explain the present device as taught. The present artincludes a device for the detection of GHB in a sample of urinesubmitted for drugs of abuse testing the steps comprise of preparing adry chemistry test means by successively impregnating a solid, carriermatrix with reagent solutions containing an indicator and a buffer, anddrying the impregnated, solid carrier matrix. Finally, by dipping saiddry chemistry test means into urine, one can observe the detectableresponse in the form of a color developed in the presence or absence ofGHB. This present art also illustrates a unique device that will preventcross contamination (runover) of test pads on the same dipstick, as wellas a unique dry chemistry test pad lateral flow device hybrid. Thesemethods can incorporate detectable responses in the visible color rangeto the human eye or in the visible light spectrum. These methods have awide sample choice other than urine, and can be replaced by anybiological sample including serum, whole blood, cerebral spinal fluid,gastric fluid, hair homogenates, sweat extracts, saliva or otherbiological fluid and other fluids such as water, beverages (beer, softdrinks, etc.), to include alcohol drinks.

EXAMPLE 4

[0134] This is a method for manufacturing a liquid, carrier-free reagentfor the adulteration detection of GBL in samples submitted for drugs ofabuse analysis.

[0135] Prepare a Solution Containing:

[0136] Solution I (R1)

[0137] Hepes buffer 2.97 g

[0138] NAD 0.3 M

[0139] gamma-butyrolactone dehydrogenase 5 mM distilled water added tomake 1000 mL total volume of solution

[0140] lab notes:

[0141] a) pH the solution to a value between 1.0 and 12.5 preferably 6.5

[0142] b) Buffer strength is preferably 0.01 Molar or greater

[0143] c) NBT needs to be in solution at a concentration 0.001 mg/L orgreater

[0144] GBL Calibrator Formulations

[0145] Zero (0) Calibrator:

[0146] 1 liter of 0.2 micron filtered normal human urine with noadulterants or drugs present, and 0.01% sodium azide.*

[0147] 50 μg/mL Calibrator:

[0148] 50 ug/mL GBL

[0149] 0.01 M Tris HCl

[0150] 100 mL of 0.2 micron filtered normal human urine with noadulterants or drugs present*

[0151] pH the solution to a value between 3.0 and 11.0 preferably 5.0.

[0152] lab notes:* Human urine can be substituted with distilled water,synthetic urine or other suitable solvent. The bacterial inhibitorsodium azide could be replaced with chloroamphenicol or other suitablebacterial inhibitors that would inhibit the growth of bacteria.

[0153] The reagent system of the instant invention (liquid reagent) isintended for use on any automatic chemistry analyzers with open channelcapability including Olympus series, Hitachi 700 series, Beckmans andmany others. The reagent as outlined in Example 5 is used in thefollowing manner: the one component of the reagent composition (R-1) isplaced in the reagent compartment of the analyzer; samples, calibrators,and controls are aliquoted into sample cups which are then placed on theanalyzer. An aliquot of 5 uL of each specimen is then pipetted into asingle, discrete cuvette followed by the addition of 150 uL of the firstreagent, R-1, and mixed; A first spectrophotometer reading is then takenfollowed by a second after a specified incubation period (i.e. oneminute for this example) at the specified wavelength (between 340 and800 nm). The spectrophotometer readings are then recorded. In thisinstance the assay is read at 340 nm. It is noted that at thiswavelength that the conversion (reduction) of NAD to NADH is observed.The absorbance of samples, and controls are printed and then compared tothe calibrator's absorbance. The quantitative value for GBLconcentration is then calculated. Any concentration of GBL greater than50.0 ug/mL is considered positive for the presence of GBL.

[0154] Please note if the present art is not used as illustrated thatvery significant increase in the cost of analysis, because a GC-MS assaymust then be performed to verify the presence of GBL. The GC-MS analysiscosts 100 times as much as the screen ($100 vs $1). Every additionalunnecessary GC-MS performed drives up the overall cost of drug testing.Eliminating these additional, unnecessary assays will save millions ofdollars per year.

[0155] Specifications for running urine samples vary from instrument toinstrument. Listed below is an example of parameters for the Hitachi 700series analyzer. The settings are intended as guidelines, and are setforth with the understanding that all those skilled in the art wouldrecognize that such parameters will vary from instrument to instrument.

[0156] The suggested specifications for the Hitachi 700 series are asfollows:

[0157] Parameter Settings for the Hitachi 700 Series Test: [GBL] Assaycode: [1 POINT] [50]-[0] Sample volume: [5] [5] R1 volume [150] [100][NO] R2 volume [ 0] [100] [NO] Wavelength [0] [340] Calib. Method:[Linear] [0] [0] Std. (1) Conc.-POS: [0.0]*-[1]* assigned calibratorvalue Std. (2) Conc.-POS: [50.0]-[2 ] assigned calibrator value Std. (3)Conc.-POS: [ ]-[ ] Std. (4) Conc.-POS: [ ]-[ ] Std. (5) Conc.-POS: [ ]-[] Std. (6) Conc.-POS: [ ]-[ ] SD Limit: [999] Duplicate Limit: [32000]Sensitivity Limit: [0] ABS. Limit (INC/DEC): [32000] [INCREASE] ProzoneLimit: [0] [lower] Expected Value: [0.0]-[1.0] Tech. Limit: [0]-[1000]Instrument Factor [1.0]

[0158] Thus as described above, an unknown urine submitted for drugs ofanaylsis for GBL will produce a value of less than the 0.0 ug/mL if noGBL is present. Conversely, if the sample has a concentration of greaterthan 50.0 ug/mL than the sample is positive for GBL.

[0159] To summarize more specifically Example 5, the automated methodfor the detection of adulteration of an unknown sample of urinesubmitted for drugs of abuse testing comprising the steps of placingaliquots of an unknown urine (or other biological sample i.e. serum,whole blood, cerebral spinal fluid, gastric fluid, hair homogenates,sweat extracts, saliva or other biological fluid and other fluids suchas beverages, water, etc.) and calibrator to be tested in automatedanalyzer sampling cups, placing the cups in a sampling tray within anautomated analyzer, transferring the aliquots of sample and calibratorto cuvettes mounted within the automated analyzer, injecting a firstreagent composition (R-1) comprising an indicator and buffer in anaqueous medium into the cuvettes, and mixing sample and reagents, andreading absorbance values of reaction mixture composed of reagents andtest samples (said test samples include urine specimens, controls, andcalibrator) at specified intervals, in accordance with a preprogrammedcode introduced into the automated analyzer, at a preprogrammedmonochromatically specified wavelength, and comparing absorbance of thefirst reagent composition plus the unknown samples with that of thefirst reagent composition plus the calibrator containing a zeroreference point (normal urinary matrix), and thereby determiningquantitatively the presence or absence of GBL.

[0160] The following changes to the above reagent solutions will remainwithin the scope and function of this invention and will have similarresults to the example above. The indicator in the solution 1, GBL,which is the indicator reactive compound (reactive in this sense meansthat gamma-butyrolactone dehydrogenase is sensitive the presence of GBLand will react with GBL during which NAD is reduced to NADH) in solution1 is gamma-butyrolactone dehydrogenase, which could be substituted withone or more of the following compounds including NAD (nicotinamideadenine dinucleotide)⁺, oxidoreductase, anti-GBL, alpha-nicotinamideadenine dinucleotide phosphate, beta-nicotinamide adenine dinucleotidephosphate, ketone group sensitive indicators and all analogs of theafore mentioned.

[0161] The Hepes buffer in solution 1, may be substituted with one ormore of the following buffers: citrate, borate, borax, sodiumtetraborate decahydrate, sodium perchlorate, sodium chlorate, sodiumcarbonate, MES (2-[N-Morpholino]ethanesulfonic acid), BIS-TRIS(bis[2-Hydroxyethyl]iminotris[hydroxymethyl]methane;2-bis[2-Hydroxyethyl]amino-2-[hydroxymethyl-1,3-propanediol), ADA(N-[2-Acetamidol]-2-iminodiacetic acid; N-[Carbaoylmethyl]iminodiacetcacid), ACES (2-[(2-Amino-2-oxoethyl)amino]ethanesulfonic acid;N-[2-Acetamido]-2-aminoethanesulfonic acid), PIPES(PiperazineN-N′-bis[2-ethanesulfonic acid)]; 1,4-Piperzinedethanesulfoicacid), MOPSO (3-[N-Morpholinol]-2-hydroxypropanesulfonic acid), BIS-TRISPROPANE (1,3-bis[tris(Hydroxymethyl)methylamino]propane), BES(N,N-bis[2-Hydroxyethyl]-2-aminoethanesulfonic acid;2-bis(2-Hydroxyethyl)amino]ethanesulfonic acid), MOPS(3-[N-Morpholino]propanesulfonic acid), TES(N-tris[Hydroxymethyl]methyl-2-aminomethanesulfonic acid;2[2-Hysroxy-1,1-bis(hydroxymethyl)-ethyl]amino)ethanesulfonic acid),TRIS (Tris[hydroxymethyl]aminomethane, DIPSO(3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesulfonic acid), TAPSO(3-[N-tris(Hydroxyethyl)methylamino]-2-hydroxypropanesulfonic acid),HEPPSO (N-[2-Hydroxythyl]piperazine-N′-[2Hydroxypropanesulfonic acid]),POPSO (Piperazine-N,N′-bis[2-hydroxypropanesulfonic acid]), EPPS(N-[2-Hydroxyethyl]piperazine-N′-[3-propanesulfonic acid), TEA(triethanolamine), TRICINE (N-tris[Hydroxymethyl]methyllycine;N-[2-Hydroxy-1-1-bis(hydroxymethyl)etyyl]glycine), BICINE(N,N-bis[2-Hydroxyethyl]glycine), TAPS(N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid;([2-Hdroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-propanesulfonic acid),AMPSO (3-[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonicacid), CHES (2-[N-Cyclohexylamino]ethanesulfonic acid), CAPSO(3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic acid), AMP2-Amino-2-ethyl-1-propanol, CAPS (3-[cyclohexylamino]-1-propanesulfonicacid), hydrochloric acid, phosphoric acid, lactic acid, sulfuric acid,nitric acid, chromic acid, boric acid, perchloric acid, potassiumhydrogen tartrate, potassium hydrogen phthalate, calcium hydroxide,phosphate, bicarbonate, sodium hydroxide, potassium hydroxide, oxalateor succinate. Other buffers with an effective pK and pH range, andcapacity suitable for maintaining the sample-reagent mixture within therequired parameters of the assay's reaction mechanism may be added tothe above group, however acidic buffers are preferred.

EXAMPLE 5

[0162] This example will illustrate in detail the exact method formanufacturing the lateral flow GBL method. Keep in mind this methodcould be utilized for any general chemistry “test pad” or pads that arecurrently used or will be used in the art. In the case of DLFHtechnology, the manufacturing process includes impregnating onto anabsorbent, solid carrier (e.g. paper) called in this example, the “testpad”, in exactly the same manner as Example 1 with similar constituents.The test pad, once impregnated, is dried, then mounted onto a solidsupport (nitrocellulose membrane) that is capable of transporting(through lateral flow) liquid to the test pad from the point ofapplication of a test sample. In simpler terms, the device is dippedinto a liquid or the liquid sample is placed on the device at the bottomor starting point for the assay. The liquid migrates from the startingapplication point to the opposite end of the nitrocellulose lateral flowpaper, during which the test pad becomes saturated with the sample. Thereaction takes place on the test pad and color develops. The developedcolor is then compared to a color chart with known concentrations of GBLthat has the appropriate colors relative to each specific concentrationof GBL(s). For example a specific color for 0.0 ug/mL GBL, 50.0 ug/mL,100.0 ug/mL, etc., for comparison. The results are then recorded. Note,the test pad must be an absorbent (wicking) material that permitsmigration of sample up the solid absorbent test pad and allows analytesand reactants to interact.

[0163] Absorbent material is successively impregnated with the followingsolutions and dried at 25 degree C.:

[0164] Solution 1

[0165] NAD 0.3 M

[0166] gamma-butyrolactone dehydrogenase 5 mM

[0167] Phosphate buffer 0.01M

[0168] NBT (nitro blue tetrazolium) 0.01 mg/L

[0169] distilled water added to 100 mL total volume of solution

[0170] pH the solution to a value between 1.0 and 12.5 preferably 8.0

[0171] lab notes: Buffer strength is preferably 10 mM or greater

[0172] In this example, the lateral flow device is prepared inaccordance with the instant invention. The lateral flow device iscomprised of a paper carrier matrix (S&S, 593 grade filter paper)impregnated with the compositions of solution 1. The paper is then cutinto test pads 5 mm by 5 mm. Note that said concentrations of any of theabove constituents can be varied to suit the DLFH lateral flow/dipstickdevice format (e.g. dependent upon paper type, and inclusion ofsemi-permeable membranes or other innovations utilized in dry chemistrytechnology). The paper is then dried using forced air. The driedimpregnated test pad is then placed at approximately 35 mm (in themiddle) of a 5 mm wide by 70 mm long nitrocellulose membrane (S&SFastTrack™ NC) and makes fluid contact with nitrocellulose lateral flowpaper. The nitrocellulose membrane is capable of transporting a liquidby capillary action or wicking from one end of the lateral flow deviceto the other in approximately 60 seconds. In this example, the DLFH hasthe dimensions of 5 mm wide by 70 mm long and can be backed by or incontact with strips of glass fiber filter material (e.g. S&S 30 grade)to aid in controlling the wicking action, or other solid supportmaterial can be used.

[0173] Again, to completely illustrate the present device the startingpoint or origin at which the sample is placed on the test device is 5 mmfrom one end of the strip, and 35 mm from the site of where the test padis placed in fluid contact with the strip. For simplicity, this examplewill have the 5 mm by 5 mm impregnated test pad placed on top of thelateral flow paper and thus be in fluid contact with the said paper.

[0174] The mechanics of how the present art's LFD and dipstick test padhybrid may be explained is as follows. The starting point or origin atwhich the sample is placed on the test device is 5 mm from one end ofthe strip, and 35 mm from site where the chemically impregnated test padis in fluid contact with the lateral flow paper. The test pad can beplaced on top of the lateral flow paper making fluid contact with thelateral flow paper from the bottom side of the test pad, or the lateralflow paper can touch the paper from the side of the test pad and remainin fluid contact with the test pad. Or the lateral flow paper can reston top of the edge of test pad or be attached and in fluid contact withthe test pad in some other manner. One of the novel advantages in usinga hybrid device made of lateral flow material and a dry chemistry testpad is the lack of cross contamination from one pad to the next fromexcessive fluid, as is inherent in the prior art. For illustration,currently there are available many different types of dry chemistry teststrips available, such as the Miles Laboratories, Inc. MULTISTIX®. Thisdevice and many other like it has multiple reagents test pads withdifferent chemistries impregnated onto each pad on a single supportmembrane backing (usually plastic). Because of the relative proximity ofthese pads to each other on the same device it is easy for crosscontamination to occur, causing unreliable results. This is called“runover” (i.e. when a reagent from one pad runs over another adjacenttest pad). The present arts eliminates runover. The applicants novelapproach to the solution of runover has not been taught prior to thepresent art and is the result of extensive research and development.

[0175] Result interpretation can be explained as follows. If the sampleis positive, with a concentration of 50.0 ug/mL GBL or more, thefollowing occurs. A drop of urine (approximately 50 uL) is applied atthe starting point or origin of the strip. The urine then migrates tothe opposite or terminal end of the strip. As the urine migrates acrossthe lateral flow material (nitrocellulose) and comes into contact withthe test pad (filter paper), the urine will saturate the pad and cause achemical reaction between the impregnated chemicals and GBL in theurine. A blue color will develop on the test pad indicating a positive(greater than 50.0 ug/mL GBL) for the presence of high levels of GBL.This color can then be compared to a color chart showing the differentcolors from colorless (white background)) to a dark blue depending uponthe concentration of the GBL(s), if greater than 50.0 ug/mL. Thereaction on the test pad is immediate thus the test results can beobserved immediately.

[0176] If the sample is negative, with a concentration of less than 50.0ug/mL of GBL present the following occurs. A drop of urine(approximately 50 uL) is applied at the starting point or origin of thestrip. The urine then migrates to the opposite or terminal end of thestrip. As the urine migrates across the lateral flow material and comesinto contact with the test pad, the urine will saturate the pad andcause a chemical reaction between the impregnated chemicals and GBL.However, this example is for a negative result, thus, no reaction occursand no color develops, indicating a negative result. This negativeresult color can then be compared to a color chart showing the differentcolors from no color developed (negative) to dark blue depending uponthe concentration of the GBL, if greater than 50 ug/mL GBL. The reactionon the test pad is immediate thus the test results can be observedimmediately.

[0177] Changes to the above reagent solution of example 6 can be madeand still remain within the scope and function of this invention andwill have similar results to examples 3 and 4 above. The indicator(s)and buffer(s) of example 5 can be replaced by all the examples andpossible substitutions as illustrated in example 3 and 4.

[0178] This brief description of the present art illustrates acompletely enabled device that would allow an individual, physician,patient, and/or technician to quickly and easily determine the presenceof the GBL in urine, providing a much needed advancement the art of GBLtesting.

[0179] To briefly explain the present device as taught. The present artincludes a device for the detection of GBL in a sample of urinesubmitted for drugs of abuse testing the steps comprise of preparing adry chemistry test means by successively impregnating a solid, carriermatrix with reagent solutions containing an indicator and a buffer, anddrying the impregnated, solid carrier matrix. Finally, by dipping saiddry chemistry test means into urine, one can observe the detectableresponse in the form of a color developed in the presence or absence ofGBL. This present art also illustrates a unique device that will preventcross contamination (runover) of test pads on the same dipstick, as wellas a unique dry chemistry test pad lateral flow device hybrid. Thesemethods can incorporate detectable responses in the visible color rangeto the human eye or in the visible light spectrum. These methods have awide sample choice other than urine, and can be replaced by anybiological sample including serum, whole blood, cerebral spinal fluid,gastric fluid, hair homogenates, sweat extracts, saliva or otherbiological fluid and other fluids such as water, beverages (beer, softdrinks, etc.), to include alcohol drinks.

EXAMPLE 6

[0180] This is a method for manufacturing a dry chemistry dipstick (teststrip) with a solid carrier for the GHB detection of in urine samplessubmitted for drugs of abuse analysis. Filter paper is successivelyimpregnated with the following solutions and dried at 25 degree C.:

[0181] Solution I

[0182] EPPS (N-[2-Hydroxyethyl]piperazine-N′-[3-propanesulfonic acid),buffer 0.97 g

[0183] distilled water added to 100 mL total volume of solution

[0184] pH the solution to a value between 1.0 and 12.5 preferably 8.0

[0185] lab notes: Buffer strength is preferably 0.01 Molar or greater

[0186] Solution 2

[0187] esterase 0.01 g/L

[0188] thymol blue 0.01 g/L

[0189] distilled water added to make 1000 mL total volume of solution

[0190] lab notes: esterase needs to be in solution at a concentration0.001 g/L or greater

[0191] In this example a dipstick was prepared in accordance with theinstant invention. The device comprised a paper carrier or solid matrixincorporated with the composition of solutions 1 and 2 above. Note thatthe concentrations of any of the following examples can be varied tosuit the dipstick device format (dependent upon paper type, or use ofsemi-permeable membrane or other suitable material). This example iscarried out using the following procedure. To produce the test means, apiece of Whatman 3 MM filter paper having approximate dimensions of 1inch by 3 inches was impregnated with solution 1 by immersing the paperinto solution 1. The paper was then dried by using forced air notexceeding 25° C. A second piece of Whatman 1 MM filter paper havingapproximate dimensions of 1 inch by 3 inches was impregnated withsolution 2 by immersing the paper into solution 2. The paper was thendried by using forced air not exceeding 25° C. The dried papers are thenlaminated to each other by the use of a non-reactive (neutral adhesive).The dried, laminated papers are then applied to one side of a piece ofdouble-sided adhesive transfer tape commercially available from 3MCompany, St. Paul, Minn. 55144. The laminate is then slit into portionsmeasuring 3 inches by 0.2 inches. One portion is then attached, via theunused adhesive side to a polystyrene sheet measuring about 1.5 inchesby 3 inches and the resulting laminate is slit parallel to its shortdimension to form test devices comprising a 1.5 inch oblong polystyrenestrip carrying a square of the impregnated papers at one end, the otherend serving as the handle. When the dipstick thus obtained is dippedinto a urine submitted for drugs of abuse testing, and no green colordevelops then no GHB is present. Conversely, if any concentration of GHBis present in the urine at a 50 ug/mL or greater a green-blue color willdevelop thus confirming the presence of GHB.

[0192] In summary, Example 6 is as follows: the foregoing dry chemistrytest strip (dipstick) method for the GHB detection in a sample of urinesubmitted for drugs of abuse testing comprises the steps of preparing atest means by successively impregnating a solid, carrier matrix withreagent solutions, drying the impregnated, solid test means, thendipping said dried test means into urine, and finally observing anycolor change in the presence or absence of GHB.

[0193] The reaction as illustrated by example 6 can best be understoodas follows. When esterase reacts with GHB an acid and alcohol areproduced as by-products of the reaction. As more GHB is present, moreacid and alcohol is generated by the reaction between GHB and esterase.The detection method for this pathway can take two different directions.The change in pH of the solution can be monitored by the use of a pHindicator or the production of alcohol can be monitored. Example 7solutions 1 and 2 above illustrate the pH monitoring pathway. Example ofthe alcohol monitoring pathway will follow.

[0194] The following changes to the above reagent solutions will remainwithin the scope and function of this invention and will have similarresults to the example above. The GHB reactive indicator in the solution1, esterase, could be substituted with one or more of thehydroxybutyrate dehydrogenase, 3-hydroxybutyrate dehydrogenase,4-hydroxybutyrate dehydrogenase, carboxyl esterase, carboxylic-esterhydrolase, β-hydroxybutyrate dehydrogenase, [R]-3-hydroxybutanoate, NAD(nicotinamide adenine dinucleotide)⁺ oxidoreductase, anda-hydroxybutyrate dehydrogenase, anti-gamma-hydroxybutyrate,alpha-nicotinamide adenine dinucleotide phosphate, beta-nicotinamide7adenine dinucleotide phosphate and all analogs of the afore mentioned.

[0195] The pH indicator of solution 2, thymol blue could be replacedwith one of the following compounds such as bromcresol green, methylred, cresol red, metanil yellow, m-cresol purple, xylenol blue, thymolblue, tropeolin OO, quinaldine red, a-dinitrophenol, methyl yellow;dimethyl yellow, bromophenol blue, tetrabromophenol blue,bromochlorophenol blue, Congo red, methyl orange, p-ethoxychrysoidinehydrochloride, napthyl red, alizarin sodium sulfonate, bromocresolgreen, ?-dinitrophenol, methyl red, lacmoid, chlorophenol red, benzoylauramine G, bromocresol purple, bromophenol red, p-nitrophenol,bromthymol blue, phenol red, p-quinonemono(bis-4-oxyphenylmethide),neutral red, quinoline blue, a-naphtholphthalein, tropeolin OOO;a-napthol orange, ethyl bis(2,4-dinitrophenyl)acetate,di-o-cresolphthalide, phenolphthalein, thymolphthalein,dimethylphenolphthalein, alizarin yellow GG; salicyl yellow, alizarinyellow R, Nile blue, 2,4,6-trinitrophenylmethylnitramine, tropeolin O,triphenylrosaniline sulfonic acid (sodium or potassium salt), indigocarmine, nitrobenzene, bromcresol green, bromcresol purple,bromchlorophenol blue, brilliant yellow, brilliant blue R, brilliantcresyl blue ALD, brilliant blue G, brlliant black BN, bromthymol blue,bromphenol red, bromphenol red, bromoxylenol blue, coomasie blue,azolitmin, litmus, pyrogallosulfonphthalein, and pyrogallored-molybdate.

[0196] The EPPS buffer in solution 1, may be substituted with one ormore of the following buffers: citrate, borate, borax, sodiumtetraborate decahydrate, sodium perchlorate, sodium chlorate, sodiumcarbonate, (Tris[hydroxymethyl]aminomethane), MES(2-[N-Morpholino]ethanesulfonic acid), BIS-TRIS(bis[2-Hydroxyethyl]iminotris[hydroxymethyl]methane;2-bis[2-Hydroxyethyl]amino-2-[hydroxymethyl-1,3-propanediol), ADA(N-[2-Acetamidol]-2-iminodiacetic acid; N-[Carbaoylmethyl]iminodiacetcacid), ACES (2-[(2-Amino-2-oxoethyl)amino]ethanesulfonic acid;N-[2-Acetamido]-2-aminoethanesulfonic acid), PIPES(PiperazineN-N′-bis[2-ethanesulfonic acid)]; 1,4-Piperzinedethanesulfoicacid), MOPSO (3-[N-Morpholinol]-2-hydroxypropanesulfonic acid), BIS-TRISPROPANE (1,3-bis[tris(Hydroxymethyl)methylamino]propane), BES(N,N-bis[2-Hydroxyethyl]-2-aminoethaesulfonic acid;2-bis(2-Hydroxyethyl)amino]ethanesulfonic acid), MOPS(3-[N-Morpholino]propanesulfonic acid), TES(N-tris[Hydroxymethyl]methyl-2-aminomethanesulfonic acid;2[2-Hysroxy-1,1-bis(hydroxymethyl)-ethyl]amino)ethanesulfonic acid),DIPSO (3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesulfonic acid),TAPSO (3-[N-tris(Hydroxyethyl)methylamino]-2-hydroxypropanesulfonicacid), HEPPSO (N-[2-Hydroxythyl]piperazine-N′-[2Hydroxypropanesulfonicacid]), POPSO (Piperazine-N,N′-bis[2-hydroxypropanesulfonic acid]), TEA(triethanolamine), TRICINE (N-tris[Hydroxymethyl]methyllycine;N-[2-Hydroxy-1-1-bis(hydroxymethyl)etyyl]glycine), BICINE(N,N-bis[2-Hydroxyethyl]glycine), TAPS(N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid;([2-Hdroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-propanesulfonic acid),AMPSO (3-[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonicacid), CHES (2-[N-Cyclohexylamino]ethanesulfonic acid), CAPSO(3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic acid), AMP2-Amino-2-ethyl-1-propanol, CAPS (3-[cyclohexylamino]-1-propanesulfonicacid), Hepes, hydrochloric acid, phosphoric acid, lactic acid, sulfuricacid, nitric acid, chromic acid, boric acid, perchloric acid, potassiumhydrogen tartrate, potassium hydrogen phthalate, calcium hydroxide,phosphate, bicarbonate, sodium hydroxide, potassium hydroxide, oxalateor succinate. Other buffers with an effective pK and pH range, andcapacity suitable for maintaining the sample-reagent mixture within therequired parameters of the assay's reaction mechanism may be added tothe above group.

[0197] Now, if in example 6, solution 2 were formulated as follows, thenthe by product of the reaction (GHB and esterase) alcohol is acted uponby the alcohol oxidase. Solution 2 in this example is then used in thesame manner as solution 2 previously in example 6 and applied to thematrix as previously illustrated.

[0198] Solution 2

[0199] esterase 0.01 g/L

[0200] alcohol oxidase 0.01 mM

[0201] ABTS (2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid))0.01%

[0202] distilled water added to make 1000 mL total volume of solution

[0203] lab notes: alcohol oxidase needs to be in solution at aconcentration 0.001 mM or greater

[0204] In this reaction the alochol oxidase is specific for methanol andnot ethanol (which is the alcohol found in beverages). Methanol would bepoisonous.

[0205] Therefore, in this example a dipstick was prepared in accordancewith the instant invention. The device comprised a paper carrier orsolid matrix incorporated with the composition of solutions 1 of example6 above and the second example of solution 2 above containing thealcohol oxidase. Note that the concentrations of any of the followingexamples can be varied to suit the dipstick device format (dependentupon paper type, or use of semi-permeable membrane or other suitablematerial). This example is carried out using the following procedure. Toproduce the test means, a piece of Whatman 3 MM filter paper havingapproximate dimensions of 1 inch by 3 inches was impregnated withsolution 1 by immersing the paper into solution 1. The paper was thendried by using forced air not exceeding 25° C. A second piece of Whatman1 MM filter paper having approximate dimensions of 1 inch by 3 incheswas impregnated with solution 2 by immersing the paper into solution 2.The paper was then dried by using forced air not exceeding 25° C. Thedried papers are then laminated to each other by the use of anon-reactive (neutral adhesive). The dried, laminated papers are thenapplied to one side of a piece of double-sided adhesive transfer tapecommercially available from 3M Company, St. Paul, Minn. 55144. Thelaminate is then slit into portions measuring 3 inches by 0.2 inches.One portion is then attached, via the unused adhesive side to apolystyrene sheet measuring about 1.5 inches by 3 inches and theresulting laminate is slit parallel to its short dimension to form testdevices comprising a 1.5 inch oblong polystyrene strip carrying a squareof the impregnated papers at one end, the other end serving as thehandle. When the dipstick thus obtained is dipped into a urine submittedfor drugs of abuse testing, and no uniform green-blue color developsthen no GHB is present.

[0206] Conversely, if any concentration of GHB is present in the urineat a 50 ug/mL or greater a blue-green color will develop thus confirmingthe presence of GHB.

[0207] In summary, Example 6 is as follows: the foregoing dry chemistrytest strip (dipstick) method for the GHB detection in a sample of urinesubmitted for drugs of abuse testing comprises the steps of preparing atest means by successively impregnating a solid, carrier matrix withreagent solutions, drying the impregnated, solid test means, thendipping said dried test means into urine, and finally observing anycolor change in the presence or absence of GHB.

[0208] The reaction as illustrated by example 6 can best be understoodas follows. When esterase reacts with GHB an acid and alcohol areproduced as by-products of the reaction. As more GHB is present, moreacid and alcohol is generated by the reaction between GHB and esterase.The detection method for this pathway can take two different directions.The change in pH of the solution can be monitored by the use of a pHindicator or the production of alcohol can be monitored. Example 7solution 1 and solution 2 (containing alcohol oxidase) illustrates usingthe alcohol monitoring pathway. Alcohol oxidase in this example willproduce as a by product of the reaction, hydrogen peroxide.

[0209] The following changes to the above reagent solutions will remainwithin the scope and function of this invention and will have similarresults to the example above. The GHB reactive indicator in the solution1, esterase, could be substituted with one or more of thehydroxybutyrate dehydrogenase, 3-hydroxybutyrate dehydrogenase,4-hydroxybutyrate dehydrogenase, carboxyl esterase, carboxylic-esterhydrolase, β-hydroxybutyrate dehydrogenase, [R]-3-hydroxybutanoate, NAD(nicotinamide adenine dinucleotide)⁺ oxidoreductase, anda-hydroxybutyrate dehydrogenase, anti-gamma-hydroxybutyrate,alpha-nicotinamide adenine dinucleotide phosphate, beta-nicotinamideadenine dinucleotide phosphate and all analogs of the afore mentioned.

[0210] The alcohol reactive enzyme of solution 2, alcohol oxidase couldbe replaced with one or more of the following compounds such as alcoholdehydrogenase, NAD, NADP, or any other enzyme or antibody reactive tothe production of alcohol as a result of the interaction of esterase andGHB.

[0211] The EPPS buffer in solution 1, may be substituted with one ormore of the following buffers: citrate, borate, borax, sodiumtetraborate decahydrate, sodium perchlorate, sodium chlorate, sodiumcarbonate, (Tris[hydroxymethyl]aminomethane), MES(2-[N-Morpholino]ethanesulfonic acid), BIS-TRIS(bis[2-Hydroxyethyl]iminotris[hydroxymethyl]methane;2-bis[2-Hydroxyethyl]amino-2-[hydroxymethyl-1,3-propanediol), ADA(N-[2-Acetamidol]-2-iminodiacetic acid; N-[Carbaoylmethyl]iminodiacetcacid), ACES (2-[(2-Amino-2-oxoethyl)amino]ethanesulfonic acid;N-[2-Acetamido]-2-aminoethanesulfonic acid), PIPES(PiperazineN-N′-bis[2-ethanesulfonic acid)]; 1,4-Piperzinedethanesulfoicacid), MOPSO (3-[N-Morpholinol]-2-hydroxypropanesulfonic acid), BIS-TRISPROPANE (1,3-bis[tris(Hydroxymethyl)methylamino]propane), BES(N,N-bis[2-Hydroxyethyl]-2-aminoethaesulfonic acid;2-bis(2-Hydroxyethyl)amino]ethanesulfonic acid), MOPS(3-[N-Morpholino]propanesulfonic acid), TES(N-tris[Hydroxymethyl]methyl-2-aminomethanesulfonic acid;2[2-Hysroxy-1,1-bis(hydroxymethyl)-ethyl]amino)ethanesulfonic acid),DIPSO (3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesulfonic acid),TAPSO (3-[N-tris(Hydroxyethyl)methylamino]-2-hydroxypropanesulfonicacid), HEPPSO (N-[2-Hydroxythyl]piperazine-N′-[2Hydroxypropanesulfonicacid]), POPSO (Piperazine-N,N′-bis[2-hydroxypropanesulfonic acid]), TEA(triethanolamine), TRICINE (N-tris[Hydroxymethyl]methyllycine;N-[2-Hydroxy-1-1-bis(hydroxymethyl)etyyl]glycine), BICINE(N,N-bis[2-Hydroxyethyl]glycine), TAPS(N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid;([2-Hdroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-propanesulfonic acid),AMPSO (3-[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonicacid), CHES (2-[N-Cyclohexylamino]ethanesulfonic acid), CAPSO(3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic acid), AMP2-Amino-2-ethyl-1-propanol, CAPS (3-[cyclohexylamino]-1-propanesulfonicacid), Hepes, hydrochloric acid, phosphoric acid, lactic acid, sulfuricacid, nitric acid, chromic acid, boric acid, perchloric acid, potassiumhydrogen tartrate, potassium hydrogen phthalate, calcium hydroxide,phosphate, bicarbonate, sodium hydroxide, potassium hydroxide, oxalateor succinate. Other buffers with an effective pK and pH range, andcapacity suitable for maintaining the sample-reagent mixture within therequired parameters of the assay's reaction mechanism may be added tothe above group.

[0212] The indicator used in solution 2, ABTS(2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) is an oxygenacceptor and is sensitive to the presence of hydrogen peroxide and canbe substituted with one or more of the following: 4-aminoantipyrine(4AAP), tetramethylbenzidine (TMB), o-phenylenediamine (OPD),o-dianisidine, 5-aminosalicylic acid (5AS), 3,3′-diaminobenzidine (DAB),3-amino-9-ethylcarbazole (AEC), 4-chloro-1-napthol (4C1N), or othersuitable compound that produces an observable color for theperoxidase/peroxide reaction. Other such compounds may include, AEC(3-Amino-9-ethyl carbazole), 2-5, dimethyl-2,5-dihydroperoxyhexane,Bis{4-[N-(3′-sulfo-n-propyl)-N-n-ethyl]amino-2,6-dimethylphenyl}methane(Bis-MAPS), N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS),N-Ethyl-N-(3-sulfopropyl)-3-methoxyaniline (ADPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)aniline (ALOS),N-Ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline (MAPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS),N-Ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS),N-(3-sulfopropyl)aniline (HALPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxy-aniline (DAOS),N-Ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline (DAPS),N-Ethyl-N-(3-sulfopropyl)aniline (ALPS),N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS),N-(3-sulfopropyl)-3,5-dimethoxyaniline (HDAPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAO),N,N-Bis(4-sulfobutyl)-3,5-dimethylaniline (MADB), and pyrogallol. Also,4-aminoantipyrine can be paired with a number of compounds to create aviolet to violet-blue color complex in the presence of theperoxide/peroxidase reaction. These compounds include2,4-Dichlorophenol, N,N-Diethyl-m-toluidine, p-Hydroxybenzene Sulfonate,N,N-Dimethylaniline, 3,5-Dichloro-2-Hydroxybenzenesulfonate,2,4,6-tribromo-3-hydroxybenzoic acid, SodiumN-Ethyl-N-(3-Sulfopropyl)-m-Anisidine, hydroxybenzoic acid,4-hydroxybenzoic acid, N-Ethyl-N-(2-hydroxy-3-Sulfopropyl)-m-toluidine,AEC (3-Amino-9-ethyl carbazole), 2-5, dimethyl-2,5-dihydroperoxyhexane,Bis{4-[N-(3′-sulfo-n-propyl)-N-n-ethyl]amino-2,6-dimethylphenyl}methane(Bis-MAPS), N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS),N-Ethyl-N-(3-sulfopropyl)-3-methoxyaniline (ADPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)aniline (ALOS),N-Ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline (MAPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS),N-Ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS),N-(3-sulfopropyl)aniline (HALPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS),N-Ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline (DAPS),N-Ethyl-N-(3-sulfopropyl)aniline (ALPS),N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS),N-(3-sulfopropyl)-3,5-dimethoxyaniline (HDAPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAO),N,N-Bis(4-sulfobutyl)-3,5-dimethylaniline (MADB), and pyrogallol.Another indicator pair that may be utilized consists of3-Methyl-2-benzothiazolinonehydrazone and Dimethylaniline.

EXAMPLE 7

[0213] This is a method for manufacturing a liquid, carrier-free reagentfor the adulteration detection of GHB in samples submitted for drugs ofabuse analysis.

[0214] Prepare a solution containing:

[0215] Solution I (R1)

[0216] phosphate buffer 1.0 M

[0217] esterase

[0218] distilled water added to make 1000 mL total volume of solution

[0219] lab notes:

[0220] a) pH the solution to a value between 1.0 and 12.5 preferably 8.0

[0221] b) Buffer strength is preferably 0.01 Molar or greater

[0222] c) NBT needs to be in solution at a concentration 0.001 mg/L orgreater

[0223] Solution II (R2)

[0224] phosphoric acid 1.0 M

[0225] sodium dichromate 10.0 mg/dL

[0226] lab notes: a) pH final solution to a value of 2.0 (range of 1.014.0)

[0227] GHB Calibrator Formulations

[0228] Zero (0) Calibrator:

[0229] 1 liter of 0.2 micron filtered normal human urine with noadulterants or drugs present, and 0.01% sodium azide.*

[0230] 50 ug/mL Calibrator:

[0231] 50 ug/mL GHB

[0232] 0.01 M Sodium Borate

[0233] 100 mL of 0.2 micron filtered normal human urine with noadulterants or drugs present*

[0234] pH the solution to a value between 3.0 and 11.0 preferably 9.0.

[0235] lab notes:* Human urine can be substituted with distilled water,synthetic urine or other suitable solvent. The bacterial inhibitorsodium azide could be replaced with chloroamphenicol or other suitablebacterial inhibitors that would inhibit the growth of bacteria.

[0236] The reagent system of the instant invention (liquid reagent) isintended for use on any automatic chemistry analyzers with open channelcapability including Olympus series, Hitachi 700 series, Beckmans andmany others. The reagent as outlined in Example 2 is used in thefollowing manner: the one component of the reagent composition (R-1) isplaced in the reagent compartment of the analyzer; samples, calibrators,and controls are aliquoted into sample cups which are then placed on theanalyzer. An aliquot of 5 uL of each specimen is then pipetted into asingle, discrete cuvette followed by the addition of 150 uL of the firstreagent, R-1, and mixed; Then the second reagent, R-2 is added to thecuvettes for each sample (urine, unknown, calibrator, standard, qualitycontrol, etc.,), and mixed; then A first spectrophotometer reading isthen taken followed by a second after a specified incubation period(i.e. one minute for this example) at the specified wavelength (between340 and 800 nm). The spectrophotometer readings are then recorded. Inthis instance the assay is read at 600 nm. The absorbance of samples,and controls are printed and then compared to the calibrator'sabsorbance. The quantitative value for GHB concentration is thencalculated. Any concentration of GHB greater than 50.0 ug/mL isconsidered positive for the presence of GHB.

[0237] Please note if the present art is not used as illustrated thatvery significant increase in the cost of analysis, because a GC-MS assaymust then be performed to verify the presence of GHB. The GC-MS analysiscosts 100 times as much as the screen ($100 vs $1). Every additionalunnecessary GC-MS performed drives up the overall cost of drug testing.Eliminating these additional, unnecessary assays will save millions ofdollars per year.

[0238] Specifications for running urine samples vary from instrument toinstrument. Listed below is an example of parameters for the Hitachi 700series analyzer. The settings are intended as guidelines, and are setforth with the understanding that all those skilled in the art wouldrecognize that such parameters will vary from instrument to instrument.

[0239] The suggested specifications for the Hitachi 700 series are asfollows:

[0240] Parameter Settings for the Hitachi 700 Series Test: [GHB] Assaycode: [1 POINT] [50]-[0] Sample volume: [5] [5] R1 volume [150] [100][NO] R2 volume [150] [100] [NO] Wavelength [0] [600] Calib. Method:[Linear] [0] [0] Std. (1) Conc.-POS: [0.0]*-[1]* assigned calibratorvalue Std. (2) Conc.-POS: [50.0]-[2 ] assigned calibrator value Std. (3)Conc.-POS: [ ]-[ ] Std. (4) Conc.-POS: [ ]-[ ] Std. (5) Conc.-POS: [ ]-[] Std. (6) Conc.-POS: [ ]-[ ] SD Limit: [999] Duplicate Limit: [32000]Sensitivity Limit: [0] ABS. Limit (INC/DEC): [32000] [INCREASE] ProzoneLimit: [0] [lower] Expected Value: [0.0]-[1.0] Tech. Limit: [0]-[1000]Instrument Factor [1.0]

[0241] Thus as described above, an unknown urine submitted for drugs ofanaylsis for GHB will produce a value of less than the 0.0 ug/mL if noGHB is present. Conversely, if the sample has a concentration of greaterthan 50.0 ug/mL than the sample is positive for GHB.

[0242] To summarize more specifically Example 7, the automated methodfor the detection of adulteration of an unknown sample of urinesubmitted for drugs of abuse testing comprising the steps of placingaliquots of an unknown urine (or other biological sample i.e. serum,whole blood, cerebral spinal fluid, gastric fluid, hair homogenates,sweat extracts, saliva or other biological fluid and other fluids suchas beverages, water, etc.) and calibrator to be tested in automatedanalyzer sampling cups, placing the cups in a sampling tray within anautomated analyzer, transferring the aliquots of sample and calibratorto cuvettes mounted within the automated analyzer, injecting a firstreagent composition (R-1) comprising an GHB reactive compound and bufferin an aqueous medium into the cuvettes, mixing sample and reagent, thensecond reagent composition (R-2) comprising the alcohol indicator is anaqueous medium into the cuvettes and again the mixing of sample andreagents occurs, and reading absorbance values of reaction mixturecomposed of reagents and test samples (said test samples include urinespecimens, controls, and calibrator) at specified intervals, inaccordance with a preprogrammed code introduced into the automatedanalyzer, at a preprogrammed monochromatically specified wavelength, andcomparing absorbance of the first reagent composition plus the unknownsamples with that of the first reagent composition plus the calibratorcontaining a zero reference point (normal urinary matrix), and therebydetermining quantitatively the presence or absence of GHB.

[0243] The following changes to the above reagent solutions will remainwithin the scope and function of this invention and will have similarresults to the example above. The indicator in the solution 1, GHB,which is the indicator reactive compound (reactive in this sense meansthat esterase is sensitive (reactive) the presence of GHB and will reactwith GHB during which alcohol and acid is produced) in solution 1 isesterase, which could be substituted with one or more of the followingcompounds including hydroxybutyrate dehydrogenase 3-hydroxybutyratedehydrogenase, 4-hydroxybutyrate dehydrogenase, carboxyl esterase,carboxylic-ester hydrolase, 13-hydroxybutyrate dehydrogenase,[R]-3-hydroxybutanoate, NAD (nicotinamide adenine dinucleotide)⁺oxidoreductase, and a-hydroxybutyrate dehydrogenase,anti-gamma-hydroxybutyrate, alpha-nicotinamide adenine dinucleotidephosphate, beta-nicotinamide adenine dinucleotide phosphate and allanalogs of the afore mentioned.

[0244] The phosphate buffer in solution 1 and phosphoric acid buffer ofsolution, may be substituted with one or more of the following buffers:citrate, borate, borax, sodium tetraborate decahydrate, sodiumperchlorate, sodium chlorate, sodium carbonate, MES(2-[N-Morpholino]ethanesulfonic acid), BIS-TRIS(bis[2-Hydroxyethyl]iminotris[hydroxymethyl]methane;2-bis[2-Hydroxyethyl]amino-2-[hydroxymethyl-1,3-propanediol), ADA(N-[2-Acetamidol]-2-iminodiacetic acid; N-[Carbaoylmethyl]iminodiacetcacid), ACES (2-[(2-Amino-2-oxoethyl)amino]ethanesulfonic acid;N-[2-Acetamido]-2-aminoethanesulfonic acid), PIPES(PiperazineN-N′-bis[2-ethanesulfonic acid)]; 1,4-Piperzinedethanesulfoicacid), MOPSO (3-[N-Morpholinol]-2-hydroxypropanesulfonic acid), BIS-TRISPROPANE (1,3-bis[tris(Hydroxymethyl)methylamino]propane), TRIS, BES(N,N-bis[2-Hydroxyethyl]-2-aminoethaesulfonic acid;2-bis(2-Hydroxyethyl)amino]ethanesulfonic acid), MOPS(3-[N-Morpholino]propanesulfonic acid), TES(N-tris[Hydroxymethyl]methyl-2-aminomethanesulfonic acid;2[2-Hysroxy-1,1-bis(hydroxymethyl)-ethyl]amino)ethanesulfonic acid),HEPES (N-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]), DIPSO(3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesulfonic acid), TAPSO(3-[N-tris(Hydroxyethyl)methylamino]-2-hydroxypropanesulfonic acid),HEPPSO (N-[2-Hydroxythyl]piperazine-N′-[2Hydroxypropanesulfonic acid]),POPSO (Piperazine-N,N′-bis[2-hydroxypropanesulfonic acid]), EPPS(N-[2-Hydroxyethyl]piperazine-N′-[3-propanesulfonic acid), TEA(triethanolamine), TRICINE (N-tris[Hydroxymethyl]methyllycine;N-[2-Hydroxy-1-1-bis(hydroxymethyl)etyyl]glycine), BICINE(N,N-bis[2-Hydroxyethyl]glycine), TAPS(N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid;([2-Hdroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-propanesulfonic acid),AMPSO (3-[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonicacid), CHES (2-[N-Cyclohexylamino]ethanesulfonic acid), CAPSO(3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic acid), AMP2-Amino-2-ethyl-1-propanol, CAPS (3-[cyclohexylamino]-1-propanesulfonicacid), hydrochloric acid, phosphoric acid, lactic acid, sulfuric acid,nitric acid, chromic acid, boric acid, perchloric acid, potassiumhydrogen tartrate, potassium hydrogen phthalate, calcium hydroxide,phosphate, bicarbonate, sodium hydroxide, potassium hydroxide, oxalateor succinate. Other buffers with an effective pK and pH range, andcapacity suitable for maintaining the sample-reagent mixture within therequired parameters of the assay's reaction mechanism may be added tothe above group, however acidic buffers are preferred.

[0245] The indicator of the presence of alcohol in solution sodiumdichromate may be replaced with one or more of the following: sulfuricacid, sodium salicylate, sodium hydroxide, iodine, potassiumpermanganate or other alcohol reactive indicators or analogs of theafore mentioned.

EXAMPLE 8

[0246] The following procedure is a method for manufacturing a drychemistry test strip (DCD), for the determination of GHB in a testsample.

[0247] Filter paper is impregnated with the following solutions anddried at 25 degree C.:

[0248] Solution 1

[0249] 30.2 G PIPES (1,4-Piperazinediethanesulfonic acid)

[0250] 0.05 Units/mL beta-Galactosidase/anti-GHB (enzyme conjugated tothe anti-GHB)

[0251] add to 900 mL D.I. water, mix, adjust pH to 6.8, Q.S. to 1000 mL

[0252] Solution 2

[0253] 0.01 M 5-bromo-6-chloro-3-indoxyl-beta-D-galactopyranoside(Magenta-beta-D-Gal)

[0254] 1 mL (0.1%) DMSO

[0255] dissolve in 900.0 mL distilled water, mix, and Q.S. to 1000 mL.

[0256] In this example, a dipstick is prepared in accordance with theinstant invention as described in Example 1, however. Solution 1 and 2are both incorporated into the test device by immersing the test paperinto solution 1; drying the paper; then immersing the test paper intosolution 2; the paper is then dried by using forced air not exceeding 60degrees C. If a two-part test pad “sandwich” is used, the pad withsolution #1 must be on top and the pad with solution 2 is on the bottom.The dipstick thus obtained will produce a magenta color when exposed toGHB at a concentration of 50 ug/mL or greater. In fact, the intensity ofthe magenta color is proportional to the concentration of the GHB,present in the sample. This test device, therefore, effectivelyidentifies the presence of GHB in urine by the measurement of the GHB inthe urine sample used for illustrative purposes in this example.

[0257] To summarize Example 8 more specifically, the foregoing drychemistry test strip (DCD) method to measure the GHB concentration in aurine sample (or other matrices) for the determination of presence ofabsence of GHB using said sample, the method comprising the steps ofpreparing a test means by successively impregnating an absorbent carriermatrix with reagent solutions, drying said test means, dipping completedtest means into test sample, and determining the quantity of GHB presentin said test sample by comparing the relative intensity of the color(magenta) produced by the reaction to a color chart with color blocksreferenced to specific concentrations of GHB.

[0258] Changes to the foregoing solutions could be made and still havesimilar results. In addition, changes to the foregoing solutions or useof the identical solutions as illustrated could be used in an automatedanalyzer and produce the same results. The foregoing solutions could becombined together, or reduced to include only 1. The concentrations ofsaid constituents may also be changed and still remain within the scopeof the invention. The buffer may be replaced with any one or more ofthose constituents enumerated in Example 1.

[0259] The indicator substrate complex in the solution5-bromo-6-chloro-3-indoxyl-beta-D-galacatopyranoside, could besubstituted with one or more of the following:4-Aminophenyl-beta-D-galactopyranoside,3-indoxyl-beta-D-galactopyranoside (blue),5-Bromo-4-chloro-3-indoxyl-beta-D-galactopyranoside (blue),5-Bromo-3-indoxyl-beta-D-galactopyranoside (blue),6-chloro-3-indoxyl-beta-D-galactopyranoside (salmon),6-Fluoro-3-indoxyl-beta-D-galactopyranoside,8-Hydroxyquinoline-beta-D-galactopyranoside,5-Iodo-3-indoxyl-beta-D-galactopyranoside (purple),N-Methylindoxyl-beta-D-galactopyranoside,2-Nitrophenyl-beta-D-galactopyranoside,4-Nitrophenyl-beta-D-galactopyranoside, NaphtholAS-BI-beta-D-galactopyranoside, and 2-Naphthyl-beta-D-galactopyranoside(yellow). Fluorescent substrates may also be utilized including4-Methylumbelliferyl-beta-D-glucuronic acid. The colors noted in theparentheses are those produced in the reaction described above. Theindicator substrate used in these examples must be matched to theconformation of the galactosidase used (i.e. alpha or beta, anddextrorotorary (D) or levorotorary (L)). For example,beta-D-Galactosidase should be matched with the indicator/substrateIodo-3-indoxyl-beta-D-galactopyranoside; conversely,alpha-L-Galactosidase would be matched withIodo-3-indoxyl-alpha-L-galactopyranoside. Note that somecross-reactivity does occur between stereo-isomers and, therefore, it ispossible to substitute these compounds where appropriate.

[0260] Substitution of the beta-Galactosidase with another enzyme wouldnecessitate a change of substrate indicator complex. If anotherglycosidase was selected, it would have to be matched to the appropriatesubstrate (e.g. beta-Cellobiosidase and a cellobioside). Examples ofsubstrates for beta-D-Cellobiosidase include5-Bromo-4-chloro-3-indoxyl-beta-D-cellobioside,5-Bromo-6-chloro-3-indoxyl-beta-D-cellobioside,4-Nitrophenyl-beta-D-cellobioside, 1-Naphthyl-cellobioside, and thefluorescent indicator, 4-Methylumbelliferyl-beta-D-cellobioside.

[0261] Other glycosidases which may be substituted for Galactosidase andCellobiosidase include the alpha and beta, and D and L conformations ofthe following enzymes: Arabinosidase, Fucosidase, Galactosaminidase,Glucosaminidase, Glucosidase, Glucuronidase, Lactosidase, Maltosidase,Mannosidase, and Xylosidase. Their corresponding substrates,Arabinopyranoside, Fucopyranoside, Galactosaminide, Glucosaminide,Glucopyranoside, Glucuronic acid, Lactopyranoside, Maltopyranoside,Mannopyranoside, and Xylopyranoside may be bound to each of thefollowing color indicator groups: 5-Bromo-4-chloro-3-indoxyl,5-Bromo-6-chloro-3-indoxyl, 6-chloro-3-indoxyl, 5-Bromo-3-indoxyl,5-Iodo-3-indoxyl, 3-indoxyl, 2-(6-Bromonaphthyl), 6-Fluoro-3-indoxyl2-Nitrophenyl, 4-Nitrophenyl, 1-Naphthyl, Naphthyl AS-BI,2-Nitrophenyl-N-acetyl, 4-Nitrophenyl-N-acetyl, and 4-Methylumbelliferylmoieties.

[0262] The glycosidase enzyme conjugated to the anti-GHB in the exampleabove can also be replaced by other types of enzymes whose substratesare compatible with the indicator groups listed above. These includeesterases (e.g. Carboxyl esterase, and Cholesterol esterase), sulfatases(e.g. Aryl sufatase), and phosphatases (e.g. Alkaline phosphatase).These enzymes can utilize the indicator groups delineated above whenconjugated to the corresponding substrate. For example, Carboxylesterase and 6-chloro-3-indoxyl butyrate, and Aryl sulfatase and5-bromo-4-chloro-3-indoxyl sulfate, and Alkaline phosphatase and2-naphthyl phosphate form enzyme-substrate pairs.

[0263] Other enzymes may be conjugated to the anti-GHB, and thereforesubstituted for the species described above. This group now listed,however, must utilize a substrate that is distinct and separate from theindicator. This enzyme group may include any dehydrogenase, oxidase,hydroxylase, or oxidoreductase. Each grouping will utilize a specificindicator or group of indicators. The dehydrogenases and hydroxylaseswill utilize a co-enzyme, a color indicator and an electron carrier suchas a-NAD (a-Nicotinamide adenine dinucleotide) or NADP, however theseelectron carrier/acceptors can be replaced by the alpha or beta isomersof any one of the following substitutes: nicotinamide adeninedinucleotide, nicotinamide adenine dinucleotide 3′-phosphate,nicotinamide adenine dinucleotide phosphate, triphosphopyridine,nicotinamide 1-N1-ethenoadenine dinucleotide phosphate, nicotinamidehypoxanthine dinucleotide, nicotinamide hypoxanthine dinucleotidephosphate, nicotinamide mononucleotide, nicotinamide N1-propylsulfonate,nicotinamide ribose monophosphate, or other analogs of NAD or NADP.

[0264] Some dehydrogenases and hydroxylases and their substrate pairswhich can be used include Formaldehyde dehydrogenase and Formaldehyde,Fructose dehydrogenase and Fructose, Glucose-6-phosphate dehydrogenaseand Glucose-6-phosphate, Glucose dehydrogenase and Glucose, Glutamatedehydrogenase and Glutamate, Glycerol dehydrogenase and Glycerol,Glycerol-3-phosphate dehydrogenase and Glycerol-3-phosphate,Hydroxybutyrate dehydrogenase and Hydroxybutyrate, Hydroxybenzoatehydroxylase and 4-Hydroxybenzoate, Lactate dehydrogenase and Lactate,Leucine dehydrogenase and Leucine, Malate dehydrogenase and Malate,Mannitol dehydrogenase and Mannitol, or any other dehydrogenase orhydroxylase.

[0265] The use of oxidases to replace the glycosidase also requires aseparate indicator, and peroxidase. Some oxidases and their substratepair which can be used include Acyl-CoA oxidase and Acyl-CoA, Alcoholoxidase and Ethanol, Ascorbate oxidase and Ascorbate, Cholesteroloxidase and Cholesterol, Choline oxidase and Choline, Glucose oxidaseand Glucose, Glycerophosphate oxidase and Glycerophosphate, Xanthineoxidase and Xanthine, Uricase and Uric acid, or any other oxidase.

[0266] A few color indicators that can be utilized with peroxidaseinclude pyrogallol, ABTS (2,2′-Azinobis(3-ethylbenzthiazoline) sulfonicacid), 3,3′,5,5′-Tetramethylbenzidine, ortho-Dianisidine,3,3′-Diaminibenzidine, AEC (3-Amino-9-ethyl carbazole), 2-5,dimethyl-2,5-dihydroperoxyhexane,Bis{4-[N-(3′-sulfo-n-propyl)-N-n-ethyl]amino-2,6-dimethylphenyl}methane(Bis-MAPS), N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS),N-Ethyl-N-(3-sulfopropyl)-3-methoxyaniline (ADPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)aniline (ALOS),N-Ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline (MAPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS),N-Ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS),N-(3-sulfopropyl)aniline (HALPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS),N-Ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline (DAPS),N-Ethyl-N-(3-sulfopropyl)aniline (ALPS),N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS),N-(3-sulfopropyl)-3,5-dimethoxyaniline (HDAPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAO), andN,N-Bis(4-sulfobutyl)-3,5-dimethylaniline (MADB). An indicator pair mayalso be used. One such pair is 3-Methyl-2-benzothiazolinonehydrazone andDimethylaniline. Another pair combines 4-aminoantipyrine with a numberof compounds to create a violet to violet-blue color complex in thepresence of the peroxide/peroxidase reaction. These compounds includephenol, 2,4-Dichlorophenol, N,N-Diethyl-m-toluidine, p-HydroxybenzeneSulfonate, N,N-Dimethylaniline, 3,5-Dichloro-2-Hydroxybenzenesulfonate,Sodium N-Ethyl-N-(3-Sulfopropyl)-m-Anisidine, andN-Ethyl-N-(2-hydroxy-3-Sulfopropyl)-m-toluidine. An example of thisassay procedure would substitute glucose oxidase for galactosidase inthe antibody-enzyme conjugate in R-1; the R-2 would then contain glucoseas the substrate and ABTS (reduced) as the indicator. The R-2 would alsocontain peroxidase, because the product of the reaction between glucoseoxidase and glucose yields peroxide. The peroxidase oxidizes anyperoxide thus produced, thereby releasing an oxygen atom; this oxygen,in turn, reacts with ABTS, and converts it from the colorless, reducedform to its blue, oxidized form. The intensity of the blue colorproduced is proportional to the GHB concentration present in thespecimen. Clearly, peroxidase may be conjugated to the antibody, and theindicators noted above used with it and its substrate, peroxide.

[0267] The use of oxireductases to replace glycosidase also requires aseparate indicator including NADPH oxidoreductase and NADPH, or anyoxidoreductase. The NADPH oxireductase reduces the NADPH in the presenceof Flavin mononucleotide (FMN). This reaction may be observed visuallyby utilizing the same color indicators as delineated for thedehydrogenases, or measured spectrophotometrically at 340 nm.

[0268] The antigens used in this example and the prior examples may besubstituted with any one or more of the following anti-GHB (I or II), orany monoclonal or polyclonal antibodies of the same. All of thesereactants can be used and will produce a detectable response in thepresence of GHB.

EXAMPLE 9

[0269] The following procedure is a method for manufacturing a drychemistry test strip, (DCD) for the determination of GHB in a testsample by measurement of its GHB concentration. Filter paper isimpregnated with the following solutions and dried at 25 degree C.:

[0270] Solution 1

[0271] 2-[N-Morpholino]ethanesulfonic Acid buffer (MES) 0.1 M

[0272] anti-GHB is conjugated to horseradish peroxidase

[0273] 900 mL D.I. water, mix, adjust pH to 6.0, and Q.S. to 1000 mLwith D.I. water

[0274] Solution 2

[0275] 2-[N-Morpholino]ethanesulfonic Acid buffer 0.1 M

[0276] Tetramethylbenzidine, (TMB) 500 mg

[0277] Urea-Peroxide, 5.0 g

[0278] 900 mL D.I. water, mix, and adjust pH between 5.0 and 7.0,preferably 6.0

[0279] Q.S. to 1000 mL with D.I. water

[0280] lab note: the techniques for producing these types of conjugatedantibodies is well known in the art.

[0281] This assay utilizes an antigen/antibody reaction with theantibody conjugated to peroxidase. When antibody which is conjugated tothe peroxidase binds to its target antigen, it releases the peroxidasewhich is then free to react with peroxide and the chromogen, TMB,resulting in formation of a blue-green colored complex. This colorreaction yields a visible color change. Therefore, the GHB concentrationis proportional to the intensity of the blue-green color produced.

[0282] The test device in this example is manufactured in the samemanner as that in Example 9. If this device is constructed using tworeaction pads, the reaction pad containing solution 2 must be on thebottom half of the “sandwich”. In addition, it may be necessary toseparate the two pads with a semipermeable membrane.

[0283] Changes to the foregoing solutions could be made and still havesimilar results. In addition, changes to the foregoing solution or useof the identical solutions could be utilized in an automated analyzerand produce the same results. The foregoing solutions could be combinedtogether, or reduced to only 1. The concentrations of said constituentsmay also be changed and still remain within the scope of the invention.Obviously, the same substitution groups for anti-GHB are possible asalready demonstrated in examples 1-9 and this includes the buffers asnoted in the prior examples also apply to this example. The ureaperoxide was chosen, because it is more stable than simple peroxide. Itis obvious, however, that one may utilize any peroxide-containingcompound to act as a substrate to peroxidase.

[0284] The TMB may be replaced by any suitable compound that willproduce an observable color as part of the peroxidase/peroxide reaction.Other such compounds include ABTS(2,2′-Azino-di-(3-ethylbenzthiazolinesulfonic acid) diammonium salt, AEC(3-Amino-9-ethyl carbazole), 2-5, dimethyl-2,5-dihydroperoxyhexane,Bis{4-[N-(3′-sulfo-n-propyl)-N-n-ethyl]amino-2,6-dimethylphenyl}methane(Bis-MAPS), N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS),N-Ethyl-N-(3-sulfopropyl)-3-methoxyaniline (ADPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)aniline (ALOS),N-Ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline (MAPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS),N-Ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS),N-(3-sulfopropyl)aniline (HALPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS),N-Ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline (DAPS),N-Ethyl-N-(3-sulfopropyl)aniline (ALPS),N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS),N-(3-sulfopropyl)-3,5-dimethoxyaniline (HDAPS),N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAO),N,N-Bis(4-sulfobutyl)-3,5-dimethylaniline (MADB), and pyrogallol. Also,4-aminoantipyrine can be paired with a number of compounds to create aviolet to violet-blue color complex in the presence of theperoxide/peroxidase reaction. These compounds include2,4-Dichlorophenol, N,N-Diethyl-m-toluidine, p-Hydroxybenzene Sulfonate,N,N-Dimethylaniline, 3,5-Dichloro-2-Hydroxybenzenesulfonate, SodiumN-Ethyl-N-(3-Sulfopropyl)-m-Anisidine, andN-Ethyl-N-(2-hydroxy-3-Sulfopropyl)-m-toluidine. Another indicator pairthat may be utilized consists of 3-Methyl-2-benzothiazolinonehydrazoneand Dimethylaniline.

[0285] In addition, it is possible to conjugate other enzymes toantibodies or antigens. Consequently, these conjugated pairs can also besubstituted into the test reaction together with an appropriateindicator compound. Therefore, this assay may include any enzyme capableof being conjugated to an antibody or antigen.

[0286] To further describe the preferred test method for determining thepresence of GHB by the measurement in an unknown test sample, the assaysystem can take the form of a dipstick (DCD), lateral flow device (LFD),or an aqueous liquid reagent that is composed of a buffer and anindicator that produces a color or change in the intensity of color orabsorbance in the UV or visible spectrum in the presence of GHB. Theantibodies (such as anti-GHB, anti-anti-GHB, anti-IgG or others). Theanti-GHB antibodies can also include IgA, IgD, IgE, and IgM. The buffersused may be any one or more compounds selected from the following groupand enumerated by their common names: citrate, hepes, tris (trizma),taps, popso, tes, pipes, mopso, tricine, mops, mes, bicine, bes, caps,epps, dipso, ches, capso, ampso, aces, ada, bis-tris-propane, tapso,heppso, tea, amp, phosphate, phthalate, succinate, hydrochloric acid,sulfuric acid, nitric acid, acetic acid, sodium hydroxide, and potassiumhydroxide. In addition, as taught the test sample can be any biologicalfluid from the following group: urine, serum, whole blood, saliva,cerebral spinal fluid, gastric contents, and extracts of hair or sweat.This art as taught herein can employ an aqueous-based liquid reagent formeasuring the concentration of GHB, said test method comprising thesteps of placing the reagent in the reagent compartment of the chemistryautoanalyzer, aliquoting samples, calibrators, and controls into samplecups and placing them on the chemistry autoanalyzer, transferring analiquot of each sample, calibrator, and control into single, discretecuvettes mounted within the chemistry autoanalyzer, aliquoting aspecified volume of the reagent composition into each cuvette andmixing, incubating the reaction mixture for a specified time interval,measuring and recording absorbance values of the reaction mixtures withthe chemistry autoanalyzer's spectrophotometer at the specifiedwavelength (from 340 to 800 nm) at preprogrammed time intervals, andcomparing absorbance values of samples and controls to those of thecalibrators in the form of a standard curve thereby quantitating the GHBif present.

[0287] This art as taught in previous examples can also employ a drychemistry test strip (DCD) method for measuring the GHB concentration ina test sample, the method comprising the steps of preparing a test meansby successively impregnating a carrier matrix with reagent solutions,drying said test means, dipping completed test means into test sample,and determining the quantity of GHB present in said test sample bycomparing the relative intensity of the color produced by the reactionto a color chart with color blocks referenced to specific concentrationsof GHB.

[0288] This art as taught in previous examples can also employ a drychemistry, lateral flow device (LFD) for measuring the GHB concentrationin a test sample, the method comprising the steps of preparing a testmeans by successively impregnating a solid, absorbent carrier matrixwith liquid, reagent solutions at specific locations on the test means,drying said test means, dipping completed test means into test sample orpipetting test sample onto the test means, and determining the quantityof GHB in the test sample by comparing the relative intensity(completeness) of color developed to a standard chart.

EXAMPLE 10

[0289] The following procedure is a method for manufacturing a drychemistry, lateral flow test strip for the determination of GHB. Thisexample will also illustrate the utility of incorporating the use ofcreatinine concentration (as determined by colorimetric assay, DCD, LFD,antibody/antigen, etc. . . . ) on the same sample measured for GHB andthe enhanced clinical significance of the GHB value. Absorbent materialis successively impregnated with the following solutions and dried at 25degree C.:

[0290] Solution 1

[0291] 0.05 M Phosphate buffer pH 7.2

[0292] 30 fmol/L anti-GHB

[0293] Solution 2

[0294] 0.05 M Phosphate pH 7.2

[0295] 30 fmol/L 50 ug/mL of GHB bound to red microparticles

[0296] In this example, the lateral flow device is prepared inaccordance with the instant invention. The lateral flow device iscomprised of a paper carrier matrix impregnated with the compositions ofsolutions 1 and 2 as follows. Note that said concentrations of any ofthe above constituents can be varied to suit the lateral flow/dipstickdevice format (e.g. dependent upon paper type, and inclusion ofsemi-permeable membranes or other innovations utilized in dry chemistrytechnology). Production of this test device is carried out using thefollowing procedure. The test device made up of a solid support whichincludes an absorbent material capable of transporting a liquid bycapillary action or wicking (e.g. nitrocellulose 5.0u, S&S brand) inthis example having dimensions of 5 mm by 70 mm and can be backed by orin contact with strips of glass fiber (e.g. Whatman GF/A) to aid incontrolling the wicking action. In this example, the device uses an GHBcutoff of 50 ug/mL.

[0297] The starting point or origin at which the sample is placed on thetest device is 5 mm from one end of the strip, and 30 mm from thisorigin is the buffered solution 1 containing anti-GHB bound to the teststrip 35 mm from the bottom edge of said test pad in a lineapproximately 1 mm wide by 5 mm long thereby extending from one side ofthe device to the other side forming the A, “assay line”. A secondbuffered solution consisting of red colored particles bound (i.e.irreversibly coupled, conjugated, or covalently linked) to 50.0 ug/mLGHB is applied to the strip approximately 5 mm from the starting point(or 10 mm from the lower edge of the test strip) in a concentration asto make certain that assay line forms a solid visual line to achieveeffective results.

[0298] A solid case made of plastic or other suitable material may beused to conceal and protect the device except for a assay line “window”(hole in the device for viewing the results of the analysis); and awindow for sample application at the origin. This case may be composedof plastic, wood, cardboard, or other suitable material.

[0299] If the sample is positive, with a concentration of 50 ug/mL GHBor more the following occurs. A drop of urine (approximately 50 uL) isapplied at the starting point or origin of the strip. The urine thenmigrates to the opposite or terminal end of the strip. The free GHBpresent (in a concentration of 50 ug/mL or greater of GHB) in the urinestarts to migrate to the assay line and binds all of the bound anti-GHBat the assay line. The red particles bound with GHB will migrate withthe urine toward the terminal end of the strip away from the startingpoint. These colored complexes will not bind to the line of anti-GHBbound at the 10 mm “A” line or assay window because all of the free GHBhas already bound up all of the active sites of the bound (immobilized)anti-GHB at the assay line. The migrating red colored particlecomplexes, therefore, continue migrating up the device and disappearfrom view. Thus, no solid red line is formed indicating that aconcentration of 50 ug/mL or more of GHB is present.

[0300] If the sample is negative, with a concentration of less than 50ug/mL GHB present, the following occurs. The free (unbound) red GHBmicroparticles complexes migrate up to the “A” assay line and bind tothe anti-GHB conjugated (immobilized) to the test strip at that locationthereby forming a solid (complete) red line assay line. The formation ofa solid red line indicates a negative for the presence of GHB inconcentration of greater than 50 ug/mL.

[0301] The test strip can be placed on top of, or backed, with glassfiber (e.g. Whatman GF/A) in order to control (i.e. speed up, or slowdown the “wicking” speed) and held in place by an adhesive or othermeans. This brief description of the present art illustrates acompletely enabled device that would allow a physician, patient, and/ortechnician to determine rapidly the presence or absence of GHB in aurine or other suitable fluid.

[0302] If analysis is performed on a 24 hour urine collection, nofurther analysis is required. Proper 24 hour urine collections aredifficult and inconvenient for the patient, however, the above test canalso be performed using a random specimen. Consequently, a noveladdition to further improve the ease of use and the accuracy of thepresent device requires an additional assay on the same random or spoturine used for the GHB assay. This additional assay is for creatinine,cystatin C or any other steady state marker consistently excreted inhuman urine. This analyte value can be used to “normalize” or correctthe GHB test result for the amount of water present in the sample. Watercontent of a random urine sample is affected by the diurnal variations,diet, diuretics (e.g. caffeine, sugar, etc. . . . ) and short term fluidconsumption (water consumed over the previous 2 to 3 hours). The amountof creatinine excreted by a normal, healthy individual is relativelyconsistent from day to day, and hour to hour; any GHB would also beexcreted at a consistent rate from hour to hour. Creatinine or CystatinC is, therefore, ideal for adjusting or normalizing the amount of GHBfound in a random urine. Specifically, if for example the creatinineconcentration is high the subject has consumed very little water overthe previous few hours, and the GHB value will be elevated; if thesubject has consumed a large volume of water just prior to testing, thecreatinine value will be low and the GHB marker will also be depressed.

[0303] This present art incorporates the unique invention of the GHBanalysis and determining the concentration of a steady state marker suchas creatinine or Cystatin C ratio (GHB/creatinine). The followingformula may be used to adjust the GHB value according to the creatinineconcentration, and thereby produce the GHB/creatinine ratio (i.e. G/Cratio). This method requires division of the GHB value by the creatinineconcentration of the sample. This yields a normalized GHB value for arandom sample. The method of measuring creatinine in urine by LFD ishitherto unknown in the art until the present device and examples ofthis methodology will follow. If analysis is being performed viaautomated chemistry, a number of well known methods are currentlyavailable. This ratio provides the most convenient way to normalize theGHB value and allow the user, even an untrained one, to obtain acorrected GHB value.

[0304] The following is a detailed description of how the GHB/creatinineratio is used. Obviously, in the case of testing the sample withaqueous, liquid reagents on an automated chemistry analyzer systemquantitative results would be obtained for both analytes. The GHB valueis then divided by the creatinine concentration. If this ratio is equalto, or greater than 0.27, then GHB presence is confirmed. Values lowerthan 0.27 are considered negative for GHB for this example.

[0305] In the example above, the device detects 50 ug/mL of GHB or morein the urine, so positives are considered 50, and negatives are zero.Typical creatinine values range from 45 to 180 mg/dl. Therefore, if theGHB result is positive and the creatinine value is less than 185 mg/dl,then the corrected result is still positive (50/185=0.27); the ratio isinversely proportional to the creatinine value (i.e. as the creatininedrops, the ratio increases). Obviously the higher the ratio, the moreGHB present. Therefore, a semi-quantitative GHB/creatinine ratio can beobtained by assuming any positive is 50 ug/mL of GHB and dividing it bythe creatinine quantitation (e.g. 50/60=0.833 ratio). On the other hand,if the creatinine concentration is higher than 185, then the true GHBvalue may be falsely elevated, and a new sample should be tested becausethis could be interpreted as a false positive.

[0306] Conversely, if the GHB value is negative, and the creatininevalue is 157 mg/dl or higher, then the sample is clearly negative (25ug/mL/157 mg/dl=0.159). On the other hand if the creatinine value islower than 20 mg/dl creatinine the assay should be repeated. It is wellknown in the art that a creatinine of less than 20 mg/dl is a dilutespecimen and a false negative could occur with this specimen(25/20=1.25, a positive).

[0307] Another factor that can and should be taken into account iskidney function as determined by the protein/creatinine ratio. If theprotein/creatinine ratio is normal (less than 3.0, as known in the art),then the assay is not affected by the ability of the kidneys to clearcreatinine or other steady state marker such as cystatin C and allow foran accurate assessment of the urine concentration. If theprotein/creatinine ratio is greater than 3.0, then the assay can beaffected by the kidney function. The GHB/creatinine ratio may becorrected for kidney dysfunction by dividing it by theprotein/creatinine-ratio (i.e. GHB/K ratio), and determining appropriateranges. Preliminary data suggests that an GHB/K ratio of 0.05 or higheris negative, and an GHB/K ratio of less than 0.05 indicates a positivefor GHB.

[0308] To summarize Example 10 more specifically, the foregoing lateralflow/dry chemistry test strip (LFD) method for measuring the GHBconcentration in a random urine sample, the method comprising the stepsof preparing a test means by successively impregnating a solid,absorbent, carrier matrix with liquid reagent solutions at specificlocations on said test means, drying said test means, dipping completedtest means into test sample or pipetting sample onto the test means, anddetermining the quantity of GHB in said test sample by comparing therelative intensity (completeness) of the assay line produced by thereaction. Also, the assay can include the determination of creatinine todetermine the GHB/creatinine ratio (G/C ratio) to improve the validityof the test result. It is understood that the above example was purelyillustrative, and that the relative positions of the control and assaylines could be relocated without changing the spirit, scope, or intentof the instant invention.

[0309] Changes to the foregoing solutions could be made and still havesimilar results. The foregoing solutions could be combined together, orreduced to include only 1 solution for impregnation. The concentrationsof said constituents may also be changed and still remain within thescope of the invention. The antibody to GHB can be to can be replacedwith antigens in appropriate positions to make for a different formatthan explained in the example. Anti-Anti-GHB could be used which is theantibody to the GHB antibody. The foregoing was merely illustrative ofthe possibilities of this novel and unique invention. In addition,anti-GBL could be used instead of anti-GHB and GBL could be conjugatedto red-microparticles the assay would work for the detection of GBL inexactly the same manner as the GHB lateral flow method.

[0310] The buffer(s) used in example 11, may be substituted with any oneor more of the buffers as illustrated in example 1.

[0311] The colored particles used in example 10 could be replaced withparticles of any color, and made from many types of materials includingrubber, latex, plastics, synthetic solids, metals, or other suitablematerial that will form a solid platform or substrate for the covalentattachment (binding) of a reactive compound, antibody, and/or antigen toit.

[0312] This Example's formulation could also include any one or more ofthe surfactants, thickeners, or interference-removing compoundsdisclosed above in this embodiment. Optional compounds for removal ofinterfering substances include mono, di, tri, and tetra sodium salts ofEDTA or EGTA. Optional thickeners include polyvinylpyrrolidone, algin,carrageenin, casein, albumin, methyl cellulose, and gelatin inconcentrations ranging from 0.5 to 5 g. per 100 ml. Optional surfactantsmay include long chain organic sulphates or sulphonates (e.g. Brij-35,Tween 20, Triton X-100, dioctyl sodium sulphosuccinate, and sodiumlauryl sulphate).

[0313] The subject invention provides an extraordinary and novel methodfor quantitating the presence of GHB in biological specimen(s) (i.e.urine, blood, serum, saliva, hair and sweat extracts, and cerebrospinalfluid) and other fluids such as water, beverages (to include but not belimited to soft drinks, beer, mixed drinks, etc.) in order to determinethe presence of GHB.

[0314] In addition, the absolute novelty of creatinine, cystatin C, orother renal clearance marker measurement by the use of a DCD or LFD isof enormous value to medical diagnostics, enforcement agencies, andpublic safety and health organizations; its utility when applied toaqueous, liquid form and modified for use on automated clinicalchemistry analyzers is also of great value for the same reasons. All inall, the ability of the present art to analyze urine for GHB measurementvia dry chemistry dipsticks, lateral flow devices, and aqueous, liquidreagents while simultaneously enabling the user to normalize the resultswith the sample's creatinine, cystatin C or other renal clearance markerconcentration as described herein is a substantial and significantimprovement over the prior art.

[0315] To further elaborate the present art so that it is clearlyunderstood the present art is a method for determining the presence ofGHB on an unknown test sample, said test method being composed of abuffer and indicator that produces a detectable response or a change inthe absorbance or intensity of a color or line in the UV or visiblespectrum in the presence or absence of GHB. These methods can use allthe buffers, indicators, microparticles (metallic or other matrix), andcomponents as taught in examples 1 through 10. The methods in examples 1through 10 as taught can be employed as aqueous liquid reagents formeasuring the concentration of GHB on a test specimen, said test methodscomprise the steps of placing the reagent in the reagent compartment ofthe chemistry autoanalyzer and aliquoting samples, calibrators, andcontrols into sample cups and placing them on the chemistryautoanalyzer, then transferring an aliquot of each sample, calibrator,and control into single, discrete cuvettes mounted within the chemistryautoanalyzer, aliquoting a specified volume of the reagent compositioninto each cuvette and mixing, incubating the reaction mixture for aspecified time interval, and measuring and recording absorbance valuesof the reaction mixtures with the chemistry autoanalyzer'sspectrophotometer at the specified wavelength (from 340 to 800 nm) atpreprogrammed time intervals, and comparing absorbance values of samplesand controls to those of the calibrators in the form of a standard curvethereby quantitating the amount of GHB present. The methods as taughtcan also employ a dry chemistry test strip (DCD) method to measure theGHB concentration in a test sample, the method comprising the steps ofpreparing a test means by successively impregnating an absorbent carriermatrix with reagent solutions, drying said test means, dipping completedtest means into test sample, and determining the quantity of GHB presentin said test sample by comparing the relative intensity of the colorproduced by the reaction to a color chart with color blocks referencedto specific concentrations of GHB. The methods as taught can also beused for any general chemistry “test pad” or pads that are currentlyused or will be used in the art like in the DCD device a furtherintegration in a device such as the DLFH, in this advance technology,the manufacturing process includes impregnating onto an absorbent, solidcarrier (e.g. paper) called in this example, the “test pad”, in exactlythe same manner as with the DCD's with similar constituents. The testpad, once impregnated, is dried, then mounted onto a solid support(nitrocellulose membrane) that is capable of transporting (throughlateral flow) liquid to the test pad from the point of application of atest sample. In simpler terms, the device is dipped into a liquid or theliquid sample is placed on the device at the bottom or starting pointfor the assay. The liquid migrates from the starting application pointto the opposite end of the nitrocellulose lateral flow paper, duringwhich the test pad becomes saturated with the sample. The reaction takesplace on the test pad and color develops. The developed color is thencompared to a color chart with known concentrations of GHB that has theappropriate colors relative to each specific concentration of GHB. Theresults are then recorded. Note, the test pad must be an absorbent(wicking) material that permits migration of sample up the solidabsorbent test pad and allows analytes and reactants to interact. Inthis example the inventor illustrates the ability of the present deviceto use any GHB reaction indicator that will produce a detectableresponse in the presence of GHB.

[0316] The methods can also employ a dry chemistry lateral flow device(LFD) for measuring the GHB concentration in a test sample, the methodcomprising the steps of preparing a test means by successivelyimpregnating a solid, absorbent carrier matrix with liquid reagentsolutions at specific locations on said test means, drying said testmeans, dipping completed test means into test sample or pipetting testsample onto the test means, and determining the quantity of GHB presentin said test sample by comparing the relative intensity of the assayline produced by the reaction to a standard chart, or by comparing therelative intensity of the assay line produced by the reaction to thecontrol line. The method examples as taught utilizing aspectrophotometer can employ wavelengths from 340 to 800 nm.

[0317] The methods as the present art teaches can also improveanalytical value of the GHB concentration of a test sample by employingcreatinine, cystatin C, or specific gravity concentrations which can beused to normalize the sample for accurate determination of GHB. Thisnormalization of the GHB concentration requires that it be divided bythe creatinine, cystatin C, or specific gravity concentration of thesame test sample thereby yielding the GHB to creatinine, cystatin C, orspecific gravity ratio. Thus, all the methods of the present art astaught are for analyzing a sample using a dry chemistry dipstick,dipstick/lateral flow hybrid, lateral flow device, or aqueous liquidreagent to determine the concentration of GHB in an individual's randomurine sample in order to determine if the individual's has ingested GHB,and normalizing or correcting this assay value with the sample'screatinine, cystatin C, or specific gravity concentration.

[0318] Changes to the above reagent solution of example 10 can be madeand still remain within the scope and function of this invention andwill have similar results to examples 1 through 10 above. Theindicator(s) and buffer(s) of example 10 can be replaced by all theexamples and possible substitutions as illustrated in examples 1 through10. It can be noted that in any of the previous examples that thereagent solutions 1 and 2 can be combined to form one reagent solutionand still be functional.

[0319] Changes to the above compound(s) used in the example(s) can beexchanged with analogs or functional derivatives of the compound(s) andstill within the scope of the appended claims.

[0320] This brief description of the present art illustrates acompletely enabled device that would allow a physician, patient,individual, enforcement agency, bar tender, parent, and/or technician toquickly and easily determine the presence of GHB or GBL in urine,providing a much needed advancement in the art of drug testing.

[0321] To briefly explain the present device as taught. The present artincludes a device for the detection of GHB in a sample of urinesubmitted for drugs testing the steps comprise of preparing a drychemistry test means by successively impregnating a solid, carriermatrix with reagent solutions containing an indicator and a buffer, anddrying the impregnated, solid carrier matrix. Finally, by dipping saiddry chemistry test means into urine, one can observe the detectableresponse in the form of a color developed in the presence or absence ofGHB. This present art also illustrates a unique device that will preventcross contamination (runover) of test pads on the same dipstick, as wellas a unique dry chemistry test pad lateral flow device hybrid. Thesemethods can incorporate detectable responses in the visible color rangeto the human eye or in the visible light spectrum. These methods have awide sample choice other than urine, and can be replaced by anybiological sample including serum, whole blood, cerebral spinal fluid,gastric fluid, hair homogenates, sweat extracts, saliva or otherbiological fluid and other fluid to include water, soft drinks,beverages, beer, mixed drinks, drinks with alcohol, etc.

[0322] As completely described and enabled the methods for determiningthe presence or amount of gamma-hydroxybutyrate (GHB) orgamma-butyrolactone in a sample, said methods comprising contacting saidsample with an indicator which specifically binds to GHB or GBL to forman indicatorcomplex; and, measuring said indicatorcomplex to determinethe presence or amount of said GHB or GBL in said sample.

[0323] The type of sample for the methods of examples 1 through 10 canbe selected from the following group consisting of urine, serum, wholeblood, cerebral spinal fluid, gastric fluid, hair homogenates, sweatextracts, saliva, water, beverages, beer, alcoholic drinks, or softdrinks.

[0324] To further delineate the foregoing teachings the methods for thedetection of GHB or GBL in a sample can also comprise the steps ofpreparing a dry chemistry test means by successively impregnating asolid, carrier matrix with reagent solutions containing an indicator anda buffer, and drying the impregnated, solid carrier matrix, and finallyplacing sample onto said dry chemistry test means, and observing thedetectable response in the form of a color developed in the presence orabsence of GHB or GBL.

[0325] And the methods as completely taught for the detection of GHB ina sample can comprise the steps of placing aliquots of an unknown urineand calibrator to be tested in automated analyzer sampling cups, placingthe cups in a sampling tray within an automated analyzer, transferringthe aliquots of sample and calibrator to cuvettes mounted within theautomated analyzer, injecting a first reagent composition (R-1)comprising an indicator and buffer in an aqueous medium into thecuvettes, mixing sample and reagent, reading the absorbance values ofreaction mixture composed of reagents and test samples (said testsamples include unknown specimens, controls, and calibrator) atspecified intervals, in accordance with a preprogrammed code introducedinto the automated analyzer, at a preprogrammed monochromaticallyspecified wavelength, and comparing absorbance of the first reagentcomposition plus the unknown samples with that of the first reagentcomposition plus the calibrator containing a zero reference point, andthereby determining the presence or absence of GHB.

[0326] It is understood that variations or modifications in thefollowing embodiments may be made by someone skilled in the art withoutdeparting from the spirit and scope of the invention. All suchmodifications and variations are to be included within the scope of theinvention as defined in the appended claims:

[0327] Referenced U.S. Patents:

[0328] U.S. Pat. No. 4,351,899

[0329] U.S. Pat. No. 4,622,296

[0330] U.S. Pat. No. 5,032,506

[0331] U.S. Pat. No. 5,624,813

[0332] U.S. Pat. No. 5,912,139

[0333] U.S. Pat. No. 5,447,837

[0334] U.S. Pat. No. 4,301,115

[0335] U.S. Pat. No. 3,603,957

[0336] Referenced EU Patents:

[0337] 0 291194

[0338] 226 427

I claim:
 1. A method for quantitatively determining gammahydroxybutyrate comprising: contacting a sample with the mixture of anenzyme conjugated to gamma hydroxybutyrate, enzyme substrate, and abuffer; determining a color change in the mixture; and correlating acolor change in the mixture with the amount of GHB in the sample.
 2. Themethod according to claim 1 wherein the enzyme is selected from thegroup consisting of glycosidases, esterases, sulfatases, phosphatases,hydroxylases and oxidoreductases.
 3. The method according to claim 1wherein the buffer is selected from the group consisting of Hepes,citrate, borate, borax, sodium tetraborate decahydrate, sodiumperchlorate, sodium chlorate, sodium carbonate, TRIS(Tris[hydroxymethyl]aminomethane, MES (2-[N-Morpholino]ethanesulfonicacid), BIS-TRIS (bis[2-Hydroxyethyl]iminotris[hydroxymethyl]methane;2-bis[2-hydroxyethyl]amino-2-[hydroxymethyl-1,3-propanediol), ADA(N-[2-Acetamidol]-2-iminodiacetic acid; N-[Carbaoylmethyl]iminodiacetcacid), ACES (2-[(2-Amino-2-oxoethyl)amino]ethanesulfonic acid;N-[2-Acetamido]-2-aminoethanesulfonic acid), PIPES(PiperazineN-N′-bis[2-ethanesulfonic acid)]; 1,4-Piperzinedethanesulfoicacid), MOPSO (3-[N-Morpholinol]-2-hydroxypropanesulfonic acid), BIS-TRISPROPANE (1,3-bis[tris(Hydroxymethyl)methylamino]propane), BES(N,N-bis[2-Hydroxyethyl]-2-aminoethaesulfonic acid;2-bis(2-Hydroxyethyl)amino]ethanesulfonic acid), MOPS(3-[N-Morpholino]propanesulfonic acid), TES(N-tris[Hydroxymethyl]methyl-2-aminomethanesulfonic acid;2[2-Hysroxy-1,1-bis(hydroxymethyl)-ethyl]amino)ethanesulfonic acid),DIPSO (3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesulfonic acid),TAPSO (3-[N-tris(Hydroxyethyl)methylamino]-2-hydroxypropanesulfonicacid), HEPPSO (N-[2-Hydroxythyl]piperazine-N′-[2Hydroxypropanesulfonicacid]), POPSO (Piperazine-N,N′-bis[2-hydroxypropanesulfonic acid]), EPPS(N-[2-Hydroxyethyl]piperazine-N′-[3-propanesulfonic acid), TEA(triethanolamine), TRICINE (N-tris[Hydroxymethyl]methyllycine;N-[2-Hydroxy-1-1-bis(hydroxymethyl)etyyl]glycine), BICINE(N,N-bis[2-Hydroxyethyl]glycine), TAPS(N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid;([2-Hdroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-propanesulfonic acid),AMPSO (3-[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonicacid), CHES (2-[N-Cyclohexylamino]ethanesulfonic acid), CAPSO(3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic acid), AMP2-Amino-2-ethyl-1-propanol, CAPS (3-[cyclohexylamino]-1-propanesulfonicacid), hydrochloric acid, phosphoric acid, lactic acid, sulfuric acid,nitric acid, chromic acid, boric acid, perchloric acid, potassiumhydrogen tartrate, potassium hydrogen phthalate, calcium hydroxide,phosphate, bicarbonate, sodium hydroxide, potassium hydroxide, oxalateand succinate.